Human antibodies specific for interleukin 15 (IL-15)

ABSTRACT

Isolated human monoclonal antibodies which specifically bind to IL-15 (e.g., human IL-15), and related antibody-based compositions and molecules, are disclosed. The human antibodies can be produced in a transfectoma or in a non-human transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies by undergoing V-D-J recombination and isotype switching. Also disclosed are pharmaceutical compositions comprising the human antibodies, non-human transgenic animals, and hybridomas which produce the human antibodies, and therapeutic and diagnostic methods for using the human antibodies.

RELATED APPLICATIONS

This application is a divisional of continuation application Ser. No.10/379,741, entitled “Human Antibodies Specific for Interleukin 15(IL-15)”, filed Mar. 5, 2003, which will issued as U.S. Pat. No.7,724,304 on Jul. 24, 2007, which is a continuation of prior filedapplication Ser. No. 10/374,932, entitled “Human Antibodies Specific forInterleukin 15 (IL-15)”, filed on Feb. 26, 2003, which is acontinuation-in-part of issued U.S. Pat. No. 7,153,507, issued on Dec.26, 2006, entitled “Human Antibodies Specific for Interleukin 15(IL-15)”, filed on Aug. 23, 2002, which claims priority to U.S.Provisional Application No. 60/314,731 entitled “Human AntibodiesSpecific for Interleukin 15 (IL-15)”, filed on Aug. 23, 2001, thecontents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Interleukin-15 (IL-15) is a pro-inflammatory cytokine, a glycoprotein of14-15 kD. Constitutive expression has been reported in various cells andtissues including monocytes and macrophages, fibroblasts, keratinocytesand dendritic cells (Waldmann and Tagaya, 1999; Fehniger and Caligiuri,2001). The expression is upregulated under inflammatory conditions, asreported for monocytes stimulated with IFN-γ and LPS or by infectionwith viruses, bacteria or protozoans (Kirman et al., 1998; Waldmann etal., 1998; Waldmann and Tagaya, 1999; Fehniger and Caligiuri, 2001).Furthermore, in chronic inflammatory diseases such as rheumatoidarthritis, locally produced IL-15 is likely to amplify inflammation bythe recruitment and activation of synovial T-cells. This IL-15-inducedeffect has been suggested to play a pivotal role in disease pathogenesis(Kirman et al., 1998; McInnes et al., 1996; McInnes et al., 1997;McInnes and Liew, 1998; Fehniger and Caligiuri, 2001).

In vitro studies have shown that IL-15 shares several biologicalactivities with IL-2, due to shared receptor components. The IL-15receptor present on T-cells consists of an unique α-chain, IL-15Rα, butshares the β-chain and the γ-chain with IL-2R. As a consequence, bothreceptors use the same Jak/STAT-signaling elements. However, based oncomplex regulation and differential expression of IL-2 and IL-15 andtheir receptors, critical differences in the in vivo functions have beenreported (Kirman et al., 1998; Waldmann and Tagaya, 1999; Waldmann etal., 2001). It is also important to note the non-redundant role forIL-15 in natural killer (NK) cell, NK-T cell and intraepitheliallymphocyte development, survival, expansion and function (Kennedy etal., 2000; Liu et al., 2000).

McInnes and coworkers (McInnes et al., 1997; McInnes and Liew, 1998)reported the induction of TNF-α production after IL-15 stimulation inT-cells derived from rheumatoid arthritis patients. Furthermore,peripheral blood T cells activated by IL-15 were shown to inducesignificant TNF-α production by macrophages via a cell-contact-dependentmechanism. Because of the destructive role of TNF-α in rheumatoidarthritis, inhibition of this cytokine decreases disease activity(Bathon et al., 2000; Klippel, 2000; Lovell et al., 2000; Maini andTaylor, 2000).

SUMMARY OF THE INVENTION

The present invention is based on the generation and isolation, for thefirst time, of fully human monoclonal antibodies which specifically bindto human IL-15 and which inhibit the proinflammtory effects induced byIL-15, as well as the characterization of such novel antibodies and thedemonstration of their therapeutic value in treating a variety of IL-15mediated diseases. For example, as described herein, the humanantibodies have been shown to inhibit both TNFα production and T cellproliferation, both of which are integrally involved in inflammatorydisorders. Accordingly, the human antibodies of the present inventionprovide an improved means for treating and preventing such disorders(and any other IL-15 mediated disorder), attributable in part to theirunique specificity (e.g., epitope and species specificity), affinity,structure, functional activity and the fact that they are fully human,making them significantly less immunogenic and more therapeuticallyeffective and useful when administered to human patients than otherIL-15 antibodies previously generated (e.g., murine and humanizedantibodies). The present invention is also based on the discovery of newtherapeutic applications, including treatment of inflammatory diseases,such as rheumatoid arthritis, psoriasis, transplant rejections andcancers, for IL-15 inhibiting antibodies such as the human antibodiesdescribed herein.

Isolated human antibodies of the invention include a variety of antibodyisotypes, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD,and IgE. Typically, they include IgG1 (e.g., IgG1k), IgG3 and IgMisotypes. The antibodies can be full-length (e.g., an IgG1 or IgG3antibody) or can include only an antigen-binding portion (e.g., a Fab,F(ab′)₂, Fv, a single chain Fv fragment, an isolated complementaritydetermining region (CDR) or a combination of two or more isolated CDRs).

In one embodiment, the human antibodies are recombinant antibodies. In aparticular embodiment, the human antibody is encoded by human IgG heavychain and human kappa light chain nucleic acids comprising nucleotidesequences in their variable regions as set forth in SEQ ID NO:1 and SEQID NO:3, respectively, and conservative sequence modifications thereof.In another embodiment, the human antibody includes IgG heavy chain andkappa light chain variable regions which comprise the amino acidsequences shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, andconservative sequence modifications thereof.

Human antibodies of the invention can be produced recombinantly in ahost cell, such as a transfectoma (e.g., a transfectoma consisting ofimmortalized CHO cells or lymphocytic cells) containing nucleic acidsencoding the heavy and light chains of the antibody, or be obtaineddirectly from a hybridoma which expresses the antibody (e.g., whichincludes a B cell obtained from a transgenic non-human animal, e.g., atransgenic mouse, having a genome comprising a human heavy chaintransgene and a human light chain transgene that encode the antibody,fused to an immortalized cell). In a particular embodiment, theantibodies are produced by a hybridoma-referred to herein as 146B7 or bya host cell (e.g., a CHO cell) transfectoma containing human heavy chainand human light chain nucleic acids which comprise nucleotide sequencesin their variable regions as set forth in SEQ ID NOs: 1 and 3,respectively, and conservative modifications thereof. In particularembodiments, the antibodies are produced by hybridomas referred toherein as 146B7, 146H5, 404E4, and 404A8. In a preferred embodiment, theantibody specifically binds to an epitope located on the β- and/orγ-chain interacting domain of IL-15.

In another embodiment, the human antibodies of the present inventionspecifically bind to human IL-15 and inhibit the ability of IL-15 toinduce proinflammatory effects, e.g., inhibit the production of TNFαand/or inhibit the proliferation of T cells, such as PBMC or CTLL-2 Tcells, upon IL-15 binding to the IL-15 receptor. Typically, the humanantibodies bind to IL-15 with a dissociation equilibrium constant(K_(D)) of less than approximately 10⁻⁷ M, such as less thanapproximately 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower when determined bysurface plasmon resonance (SPR) technology in a BIACORE 3000 instrumentusing recombinant human IL-15 as the analyte and the antibody as theligand. In a particular embodiment, the antibody binds to human IL-15with a dissociation equilibrium constant (K_(D)) of approximately6.5×10⁻⁸ M.

In a further embodiment, the invention relates to an isolated humanmonoclonal antibody which specifically binds to human IL-15, comprisingat least one CDR sequence selected from the group consisting of:

(i) SEQ ID NOs: 5, 6, 7, 8, 9, and 10;

(ii) sequences which are at least 90% homologous, preferably at least95% homologous, and more preferably at least 98%, or at least 99%homologous to the sequences defined in (i); and

(iii) fragments of the sequences defined in (i) or (ii), which retainthe ability to specifically bind to human IL-15.

In a further embodiment, the invention relates to an isolated humanmonoclonal antibody which specifically binds to human IL-15 comprising

(i) SEQ ID NO:7;

(ii) a sequence which is at least 90% homologous, preferably at least95% homologous, and more preferably at least 98%, or at least 99%homologous to SEQ ID NO:7; or

(iii) a fragment of the sequence defined in (i) or (ii), which retainsthe ability to specifically bind to human IL-15.

In a further embodiment, the invention relates to an isolated humanmonoclonal antibody which specifically binds to human IL-15, comprising

(i) SEQ ID NOs:5 and 8;

(ii) SEQ ID NOs:6 and 9;

(iii) SEQ ID NOs:7 and 10;

(iv) sequences which are at least 90% homologous, preferably at least95% homologous, and more preferably at least 98%, or at least 99%homologous to the sequences defined in (i), (ii) or (iii); or

(v) fragments of the sequences defined in (i), (ii), (iii) or (iv),which retain the ability to specifically bind to human IL-15.

In a further embodiment, the invention relates to an isolated humanmonoclonal antibody which specifically binds to human IL-15, comprisingat least four CDRs selected from

(i) SEQ ID NOs:5, 6, 7, 8, 9, or 10;

(ii) sequences which are at least 90% homologous, preferably at least95% homologous, and more preferably at least 98%, or at least 99%homologous to the sequences defined in (i); and

(iii) fragments of the sequences defined in (i) or (ii), which retainthe ability to specifically bind to human IL-15.

In a further embodiment, the invention relates to an isolated humanmonoclonal antibody which specifically binds to human IL-15, comprising

(i) SEQ ID NOs:5, 6, 7, 8, 9, or 10;

(ii) sequences which are at least 90% homologous, preferably at least95% homologous, and more preferably at least 98%, or at least 99%homologous to the sequences defined in (i); or

(iii) fragments of the sequences defined in (i) or (ii), which retainthe ability to specifically bind to human IL-15.

In a further embodiment, the invention relates to an isolated humanmonoclonal antibody which specifically binds to human IL-15, comprisinga heavy chain variable region with amino acid sequence SEQ ID NO:2; or asequence which is at least 90% homologous, preferably at least 95%homologous, and more preferably at least 98%, or at least 99% homologouswith SEQ ID NO:2.

In a further embodiment, the invention relates to an isolated humanmonoclonal antibody which specifically binds to human IL-15, comprisinga light chain variable region with amino acid sequence SEQ ID NO:4; or asequence which is at least 90% homologous, preferably at least 95%homologous, and more preferably at least 98%, or at least 99% homologouswith SEQ ID NO:4.

In a further embodiment, the invention relates to an isolated humanmonoclonal antibody which specifically binds to human IL-15, whichinhibits cis-signalling via the IL-15Rγ-chain by specifically binding toan epitope located on the γ-chain interacting domain of human IL-15, andwhich inhibits trans-signalling on neighboring cells expressing theγ-chain or the β- and γ-chains as part of IL-15R or another cytokinereceptor.

In yet another embodiment, the isolated human monoclonal antibodyspecifically binds to human IL-15 and interferes with IL-15 receptor α-,β- and γ-chain assembly and/or inhibits assembly on neighboring cellsexpressing β- and γ-chains as part of the IL-15 receptor or anothercytokine receptor.

In another aspect, the invention provides nucleic acid moleculesencoding the antibodies, or antigen-binding portions, of the invention.Accordingly, recombinant expression vectors which include theantibody-encoding nucleic acids of the invention, and host cellstransfected with such vectors, are also encompassed by the invention, asare methods of making the antibodies of the invention by culturing thesehost cells.

The invention also relates to an expression vector comprising anucleotide sequence encoding heavy and light variable regions whichcomprise the amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4,respectively, and conservative modifications thereof. Such expressionvectors are well known in the art. Examples hereof include in vitrotranscription/translation vectors using, for example, reticulocytelysates.

In yet another aspect, the invention provides isolated B-cells from atransgenic non-human animal, e.g., a transgenic mouse, which are capableof expressing various isotypes (e.g., IgG, IgA and/or IgM) of humanmonoclonal antibodies that specifically bind to IL-15. Preferably, theisolated B cells are obtained from a transgenic non-human animal, e.g.,a transgenic mouse, which has been immunized with a purified or enrichedpreparation of IL-15 antigen and/or cells expressing IL-15. Preferably,the transgenic non-human animal, e.g., a transgenic mouse, has a genomecomprising a human heavy chain transgene and a human light chaintransgene. The isolated B-cells are then immortalized to provide asource (e.g., a hybridoma) of human monoclonal antibodies to IL-15.

Accordingly, the present invention also provides a hybridoma capable ofproducing human monoclonal antibodies that specifically bind to IL-15.In one embodiment, the hybridoma includes a B cell obtained from atransgenic non-human animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a human light chaintransgene fused to an immortalized cell. The transgenic non-human animalcan be immunized with a purified or enriched preparation of IL-15antigen and/or cells expressing IL-15 to generate antibody-producinghybridomas. Particular hybridomas provided by the invention include146B7, 146H5, 404E4, and 404A8.

In yet another aspect, the invention provides a transgenic non-humananimal, such as a transgenic mouse, which expresses human monoclonalantibodies that specifically bind to IL-15. In a particular embodiment,the transgenic non-human animal is a transgenic mouse having a genomecomprising a human heavy chain transgene and a human light chaintransgene. The transgenic non-human animal can be immunized with apurified or enriched preparation of IL-15 antigen and/or cellsexpressing IL-15. Preferably, the transgenic non-human animal, e.g., thetransgenic mouse, is capable of producing multiple isotypes of humanmonoclonal antibodies to IL-15 (e.g., IgG, IgA and/or IgM) by undergoingV-D-J recombination and isotype switching. Isotype switching may occurby, e.g., classical or non-classical isotype switching.

In another aspect, the present invention provides methods for producinghuman monoclonal antibodies which specifically react with IL-15. In oneembodiment, the method includes immunizing a transgenic non-humananimal, e.g., a transgenic mouse, having a genome comprising a humanheavy chain transgene and a human light chain transgene, with a purifiedor enriched preparation of IL-15 antigen and/or cells expressing IL-15.B cells (e.g., splenic B cells) of the animal are then obtained andfused with myeloma cells to form immortal, hybridoma cells that secretehuman monoclonal antibodies against IL-15.

In another aspect, the present invention features a human anti-IL-15antibody conjugated to a therapeutic moiety, e.g., a cytotoxic drug, anenzymatically active toxin, or a fragment thereof, a radioisotope, or asmall molecule anti-cancer drug.

In another aspect, the present invention provides compositions, e.g.,pharmaceutical and diagnostic compositions, comprising apharmaceutically acceptable carrier and at least one human monoclonalantibody of the invention which specifically binds to IL-15. Thecomposition can further include other therapeutic agents, such as otherimmunosuppressive agents, or chemotherapeutic agents.

In yet another aspect, the invention provides methods for inhibiting theproinflammatory effects of IL-15, such as inhibiting IL-15 induced TNFαproduction and/or T cell proliferation, preferably without inhibitingthe activity (e.g., TNFα production and/or T cell proliferation) ofstructurally related proteins/cytokines (e.g., IL-2) using one or morehuman antibodies of the invention.

Human antibodies of the present invention can be used to treat and/orprevent a variety of IL-15 mediated diseases by administering theantibodies to patients suffering from such diseases.

Exemplary diseases that can be treated (e.g., ameliorated) or preventedusing the methods and compositions of the invention include, but are notlimited to, inflammatory disorders, such as arthritis (e.g., psoriaticarthritis and rheumatoid arthritis including active rheumatoid arthritisand juvenile rheumatoid arthritis), inflammatory bowel disease. Forexample, the antibodies have been shown to reduce parakeratosis, reduceepidermal thickness and reduce proliferation of keratinocytes inpsoriasis. The antibodies also have been shown to reduce inflammationand/or prevent chemotaxis of activated leukocytes involved in rheumatoidarthritis. The antibodies also can be used to treat infectious diseases,such as HIV infection. Furthermore, the antibodies can be used to treattransplant rejection. Accordingly, the human monoclonal antibodies ofthe invention may be useful in patients undergoing or who have undergoneorgan or tissue transplantation, such as heart, lung, combinedheart-lung, trachea, kidney, liver, pancreas, oesophagus, bowel, skin,limb transplantation, umbilical cord transplantation, stem celltransplantation, islet cell transplantation, etc.

Antibodies of the present invention may thus be used in prophylaxis ofallograft and xenograft rejection or be used to reverse, treat, orotherwise ameliorate acute allograft or zenograft rejection episodes.

Further diseases that can be treated include graft-versus-host disease,e.g. blood transfusion graft-versus-host disease and bone marrowgraft-versus-host disease Still further, the antibodies can be used totreat a variety of diseases involving IL-15 mediated neovascularization,such as tumor growth and cancers, e.g. T-cell leukaemia. Other exampleswith increased angiogenesis include inflammatory diseases, such asrheumatoid arthritis.

In a further embodiment, the invention relates to a method of treatingor preventing a disorder that is associated with an overexpression ofhuman IL-15, and/or in which a downregulation or inhibition of humanIL-15 induced effects is beneficial, comprising administering theantibody according to the invention to a subject in an amount effectiveto treat or prevent the disorder.

In a further embodiment the disorder is selected from the groupconsisting of

arthritides, such as ankylosing spondylitis, reactive arthritis,sacroileitis, and adult Still's disease;

connective tissue disorders, such as systemic lupus erythematosus,discoid lupus, CNS lupus, lupus nephritis, sarcoidosis, CNS sarcoidosis,and polymyositis/dermatomyositis;

opthalmological disorders, such as uveitis and choreoritinitis;

neurological disorders, such as myelopathy/tropical spastic paraparesis,myasthenia gravis, cervical uterine cancer, rhabdomyosarcoma, Ewing'ssarcoma, and multiple sclerosis;

gastrointestinal and hepatic disorders, such as acute fulminanthepatitis, coeliaki, post-operative enterocolitis, ulcerative colitis,and Crohn's disease;

allergic disorders, such as bronchial asthma;

hematologic disorders, such as acute T-cell lymphoblastoid leukaemia,adult T-cell leukaemia, Sezary's syndrome, chronic lymphocyticleukaemia, mycosis fungoides, precursor B-cell acute lymphoblasticleukaemia/lymphoma, chronic myelogenous leukemia, acute myeloidleukaemia, large granular lymphocytosis, large granular lymphocyteleukaemia, myeloma, plasmacytoma, plasma cell myeloma, heavy chaindiseases (including γ, λ and α disease), extranodal naturalkiller/T-cell lymphoma, and aggressive natural killer-cell leukemia;

skin disorders, such as allergic contact excema, bullous pemphigoid,post-burn hypertrophic scars, and lichen ruber; pulmonary disorders,such as chronic obstructive lung disease, fibrosing alveolitis, andacute respiratory distress syndrome;

malignancies, such as colorectal cancer, and malignant melanoma;

transplantation-derived disorders, such as allograft and xenograftrejection, and graft-versus-host disease;

endocrinologic disorders, such as autoimmune thyroiditis and Grave'sdisease;

vascular disorders, such as Wegener's granulomatosis, microscopicpolyangiitis, polyarteritis nodosa, giant-cell arteritis, andatherosclerosis;

gynecological-obstretical disorders, such as recurrent spontaneousabortion, and endometriosis; and

infectious diseases, such as sepsis, and AIDS.

In yet a further embodiment, the disorder is selected from the groupconsisting of ankylosing spondylitis, systemic lupus erythematosus,ulcerative colitis, allograft rejection and graft-versus-host disease.

The human antibodies of the present invention may also be combined withone or more additional therapeutic agents, such as anti-inflammatoryagents, DMARDs (disease-modifying anti-rheumatic drugs),immunosuppressive agents, chemotherapeutics, and psoriasis agents.

In one embodiment, the subject can be additionally treated with one ormore agents that enhance the inhibition of the proinflammatory effect ofthe antibodies, e.g., an anti-inflammatory agent, such as a steroidaldrug or a NSAID (nonsteroidal anti-inflammatory drug). Preferred agentsinclude, for example, aspirin and other salicylates, Cox-2 inhibitors,such as rofecoxib (Vioxx) and celecoxib (Celebrex), NSAIDs such asibuprofen (Motrin, Advil), fenoprofen (Nalfon), naproxen (Naprosyn),sulindac (Clinoril), diclofenac (Voltaren), piroxicam (Feldene),ketoprofen (Orudis), diflunisal (Dolobid), nabumetone (Relafen),etodolac (Lodine), oxaprozin (Daypro), and indomethacin (Indocin).

In another embodiment, the human antibodies of the invention can beadministered in combination with one or more DMARDs, such asmethotrexate (Rheumatrex), hydroxychloroquine (Plaquenil), sulfasalazine(Asulfidine), pyrimidine synthesis inhibitors, e.g. leflunomide (Arava),IL-1 receptor blocking agents, e.g. anakinra (Kineret), and TNF-αblocking agents, e.g. etanercept (Enbrel), infliximab (Remicade) andadalimumab.

Further examples are IL-10, soluble IL-15R, anti-IL6R antibodies,CTLA4Ig, and anti-CD20 antibodies.

In another embodiment, the human antibodies of the invention can beadministered in combination with one or more immunosuppressive agents,such as cyclosporine (Sandimmune, Neoral) and azathioprine (Imural).

Further examples are mycophenolic acid, mycophenolate mofetil,corticosteroids, such as prednisone, methotrexate, gold salts,sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine,15-deoxyspergualine, 6-mercapto-purine, cyclophosphamide, rapamycin,tacrolimus (FK-506), and anti-thymocyte globulin.

In another embodiment, the human antibodies of the invention can beadministered in combination with two or more immunosuppressive agents,such as prednisone and cyclosporine; prednisone, cyclosporine andazathioprine; or prednisone, cyclosporine and mycophenolate mofetil.

In another embodiment, the human antibodies of the invention can beadministered in combination with one or more chemotherapeutics, such asdoxorubicin (Adriamycin), cisplatin (Platinol), bleomycin (Blenoxane),carmustine (Gliadel), cyclophosphamide (Cytoxan, Procytox, Neosar), andchlorambucil (Leukeran). The human antibodies according to the inventioncan also be administered in conjunction with radiation therapy.

In another embodiment, the human antibodies of the invention can beadministered in combination with one or more agents for treatingpsoriasis, such as topical medications containing coal tar, A vitamin,cortisone or other corticosteroids, oral or injected medications, suchas corticosteroids, methotrexate, retinoids, e.g. acicretin (Neogitason)or cyclosporine (Sandimmune, Neoral). Other treatments may includeexposure to sunlight or phototherapy.

Further examples are anthralin, calcipotrien, tarazotene, etanercept,alefacept, efalizumab, 6-thioguanine, mycophenolate mofetil, tacrolimus(FK-506), and hydroxyurea. Other examples are CTLA4Ig and infliximab.Other treatments may include UVB (broad-band and narrow-band ultravioletB), UVA (ultraviolet A) and PUVA (psoralen methoxalen plus ultravioletA).

In a further embodiment, the compositions of the invention areadministered in conjunction with two or more of the above therapies,such as methotrexate+phototherapy (PUVA or UVA); methotrexate+acitretin;acitretin+phototherapy (PUVA or UVA);methotrexate+acitretin+phototherapy (PUVA or UVB;hydroxyurea+phototherapy (PUVA or UVB); hydroxyurea+acitretin;cyclosporine+methotrexate; or calcipotrien+phototherapy (UVB).

In another embodiment, the human antibodies of the invention can beadministered in combination with other antibodies, such as CD4 specificantibodies and IL-2 specific antibodies. A combination of the presenthuman antibodies with CD4 specific antibodies or IL-2 specificantibodies are considered particularly useful for treating autoimmunediseases and transplant rejections.

In still another embodiment, the present antibodies may be administeredin combination with other antibodies, e.g. other immunosuppressive humanmonoclonal antibodies, such as antibodies binding to, e.g., MHC, CD2,CD3, CD7, CD28, B7, CD40, CD45, IFN-γ, TNF-α, IL-2R, IL-4, IL-5, IL-6R,IL-7, IL-8, IL-10, CD11a, CD20 or CD58, or their ligands; or otherimmunomodulatory compounds, e.g., soluble IL-15R or IL-10.

In yet another aspect, the present invention provides a method fordetecting in vitro or in vivo the presence of the IL-15 antigen in asample, e.g., to diagnose IL-15-mediated diseases. In one embodiment,this is achieved by contacting a sample to be tested, along with acontrol sample, with a human monoclonal antibody of the invention, or anantigen-binding portion thereof under conditions that allow forformation of a complex between the antibody and IL-15. Complex formationis then detected (e.g., using an ELISA) in both samples, and anystatistically significant difference in the formation of complexesbetween the samples is indicative of the presence of the IL-15 antigenin the test sample.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 includes graphs showing the binding of the human IL-15 specificantibodies, 146B7, 147H5, 404A8 and 404E4, to human IL-15 (hIL-15) andto the mutant IL-15 proteins, Q108S and D8SQ108S. Serial dilutions ofthe antibodies were examined for their binding to hIL-15 or the mutantIL-15 proteins D8SQ108S and Q108S in an ELISA.

FIGS. 2 and 3 show amino acid (SEQ ID NOs:2 and 4) and nucleotide (SEQID NOs:1 and 3) sequences of the V_(H) and V_(L)-regions, respectively,from antibody 146B7. The framework (FR) and complementarity determiningregions (CDR) are indicated.

FIGS. 4A-D include graphs showing the inhibition of IL-15-mediated TNF-αrelease by antibody 146B7. Human PBMC were incubated with hIL-15 (0, 50,100 ng/ml) in combination with 146B7 antibody or with an isotype controlantibody (0.1, 1, 10 μg/ml) for 72 hours. The amount of TNF-α producedwas measured by ELISA. Data from two healthy volunteers are shown.

FIG. 5 is a graph showing the effect of antibody 146B7 on IL-2 orIL-15-mediated TNF-α production. Human PBMC were incubated with hIL-15(0, 50, 100 ng/ml) or with hIL-2 (100 ng/ml) in combination with 146B7(0.1, 1, 10 μg/ml) for 72 hours. The amount of TNF-α produced wasmeasured by ELISA.

FIG. 6 is a graph showing the inhibitory activity of antibodies 146B7,146H5, 404E4 and 404A8 on hIL-15 induced CTLL-2 proliferation. CTLL-2cells starved for hIL-2 were incubated with hIL-15 (60 pg/ml) combinedwith serial dilutions of 146B7, 146H5, 404E4 and 404A8 for 48 hours.[³H]-Thymidine incorporation was measured to express proliferation(cpm). The results are presented as mean values.

FIGS. 7-9 include graphs showing the inhibitory activity of antibodies146B7 (FIG. 7), 404E4 (FIG. 8) and 404A8 (FIG. 9) on IL-15 induced PBMCproliferation. Human PBMC were incubated with hIL-15 (0, 25, 100 ng/ml;FIGS. 7A, 8A, and 9A, respectively) or hIL-2 (0, 10, 100 ng/ml; FIGS.7B, 8B, and 9B, respectively) in combination with 146B7 (FIG. 7), 404E4(FIG. 8) or 404A8 (FIG. 9) at 0.1, 1, 10 μg/ml for 72 hours.[3H]-Thymidine incorporation was measured to express proliferation(cpm).

FIG. 10 is a graph showing the binding of antibody 146B7 toIFNγ-stimulated monocytes. Human PBMCs were cultured in the presence ofIFNγ (500 U/ml) for up to 2 days (37° C.). Fluorescence intensity of atleast 5000 cells per sample was determined after analysis by flowcytometry and gating on the monocytes. Data show the stimulation index(S.I.=(mean fluorescence positive staining)/(mean fluorescencebackground staining)).

FIG. 11 shows binding of human monocytes with antibody 146B7 (panel B)or with the isotype control antibody (panel A). Human PBMCs wereisolated and cytospins were made after culturing the cells with IFNγ(500 U/ml). Cells were counterstained with haematoxylin.

FIG. 12 shows binding of human psoriatic skin with 146B7 (panel B) orwith the isotype control antibody (hIgG1) (panel A). Human psoriaticplaques were obtained from patients after informed consent, and storedat −80° C. until assay. Tissues were stained with biotinylatedantibodies and visualized after activation of horse radish peroxidase.

FIG. 13A is a graph showing the percentage of nucleated cells inrheumatoid arthritic tissue after treatment of SCID mice with 146B7 orwith vehicle. Tissues were stained with haematoxilin and eosin (H&E) andanalysed with Photo Shop version 6.0. Data are shown as mean and s.e.m.of nuclei (as percentage of total area) of mice after 146B7 treatment(n=4) or vehicle treatment (n=2). FIGS. 13B and 13C show arepresentative H&E staining of xenografted RA tissue in SCID mice, aftertreatment with 146B7 (FIG. 13C) or with PBS (FIG. 13B).

FIG. 14 includes graphs showing the effects of antibody 146B7 treatmentin SCID/psoriasis mice. Biopsies were fixed in formalin for paraffinembedding and stained in H&E and for Ki-67 nuclear antigen. FIG. 14Ashows the severity of psoriasis evaluated by epidermal thickness whichwas measured from the stratum corneum to the beginning of the rete pegs.FIG. 14B shows the epidermal thickness which was measured from thestratum corneum to the deepest part of the rete pegs. FIG. 14C shows thegrade of parakeratosis. FIG. 14D shows the number of inflammatorymononuclear cells in upper dermis. FIG. 14E shows the number of Ki-67+cycling keratinocytes.

FIG. 15 shows H&E staining of human psoriatic skin engrafted in SCIDmice, after treatment with antibody 146B7 (panel C), with CsA (panel B),or with vehicle (panel A). Three weeks after transplantation micereceived PBS (placebo), CsA (cyclosporine A) (Sandoz) at a dose of 10mg/kg every second day for 15 days, or 146B7 at a dose of 20 mg/kg onday 1 and 10 mg/kg on days 8 and 15. One week after the last injection,mice were sacrificed, and a 4 mm punch biopsy was taken from eachxenograft. Biopsies were fixed in formalin for paraffin embedding andstained in H&E.

FIG. 16 shows Ki-67 staining of human psoriatic skin engrafted in SCIDmice, after treatment with 146B7 (panel C), with CsA (panel B), or withvehicle (panel A). Three weeks after transplantation mice received PBS(placebo), CsA (cyclosporine A) (Sandoz) at a dose of 10 mg/kg everysecond day for 15 days, or 146B7 at a dose of 20 mg/kg on day 1 and 10mg/kg on days 8 and 15. One week after the last injection, mice weresacrificed, and a 4 mm punch biopsy was taken from each xenograft.Biopsies were fixed in formalin for paraffin embedding and stained forKi-67 nuclear antigen.

FIG. 17 is a graph showing the binding of antibody 146B7 toreceptor-bound IL-15. Plates were coated with IL-15Rα and incubated withIL-15. After 10 minutes, biotinylated 146B7 was added to the wells.Binding of 146B7 to receptor-bound IL-15 was evaluated at 405 nm in anELISA-reader.

FIG. 18 is a graph showings the binding of antibody 146B7 to IL-15,after binding of IL-15 to its receptor expressed on Raji cells. Afterincubation of IL-15R-expressing Raji cells with IL-15, biotinylated146B7 was added to the cells after 10 minutes. Binding of 146B7 toreceptor-bound IL-15 was evaluated by FACS analysis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel antibody-based therapeutics fortreating and diagnosing a variety of disorders mediated by IL-15 (i.e.,disorders caused by the proinflammatory effects of IL-15). As usedherein, the term “proinflammatory effects of IL-15” includes any humoralor cell-mediated immune response induced by IL-15, such as production ofTNFα and other inflammatory mediators, and recruitment/proliferation ofT-cells. Therapies of the invention employ isolated human monoclonalantibodies which specifically bind to an epitope present on IL-15.

In one embodiment, the human antibodies are produced in a non-humantransgenic animal, e.g., a transgenic mouse, capable of producingmultiple isotypes of human monoclonal antibodies to IL-15 (e.g., IgG,IgA and/or IgE) by undergoing V-D-J recombination and isotype switching.Accordingly, various aspects of the invention include antibodies andpharmaceutical compositions thereof, as well as non-human transgenicanimals, B-cells, host cell transfectomas, and hybridomas for makingsuch monoclonal antibodies. Methods of using the antibodies of theinvention to detect cells to which IL-15 is bound, and/or to inhibitIL-15 mediated functions either in vitro or in vivo, are alsoencompassed by the invention. Methods for targeting agents to cells towhich IL-15 is bound are also included.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “IL-15,” “IL-15 antigen” and “Interleukin 15” are usedinterchangeably herein, and include any variants or isoforms which arenaturally expressed by cells.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The V_(H) and V_(L) regions canbe further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., IL-15). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), CL and CH1 domains;(ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR) or (vii) a combination of two or more isolatedCDRs which may optionally be joined by a synthetic linker. Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies.

The term “monoclonal antibody” as used herein, refers to an antibodywhich displays a single binding specificity and affinity for aparticular epitope. Accordingly, the term “human monoclonal antibody”refers to an antibody which displays a single binding specificity andwhich has variable and constant regions derived from human germlineimmunoglobulin sequences. In one embodiment, human monoclonal antibodiesare produced by a hybridoma which includes a B cell obtained from atransgenic non-human animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further in Section I,below), (b) antibodies isolated from a host cell transformed to expressthe antibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable and constant regionsderived from human germline immunoglobulin sequences. In certainembodiments, however, such recombinant human antibodies can be subjectedto in vitro mutagenesis (or, when an animal transgenic for human Igsequences is used, in vivo somatic mutagenesis) and thus the amino acidsequences of the V_(H) and V_(L) regions of the recombinant antibodiesare sequences that, while derived from and related to human germlineV_(H) and V_(L) sequences, may not naturally exist within the humanantibody germline repertoire in vivo.

As used herein, a “heterologous antibody” is defined in relation to thetransgenic non-human organism producing such an antibody. This termrefers to an antibody having an amino acid sequence or an encodingnucleic acid sequence corresponding to that found in an organism notconsisting of the transgenic non-human animal, and generally from aspecies other than that of the transgenic non-human animal.

An “isolated antibody”, as used herein, is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to IL-15 is substantially free of antibodies thatspecifically bind antigens other than IL-15). An isolated antibody thatspecifically binds to an epitope of IL-15 may, however, havecross-reactivity to other related cytokines or to other IL-15 proteinsfrom different species. However, the antibody preferably always binds tohuman IL-15. In addition, an isolated antibody is typicallysubstantially free of other cellular material and/or chemicals. In oneembodiment of the invention, a combination of “isolated” monoclonalantibodies having different IL-15 specificities are combined in a welldefined composition.

As used herein, “specific binding” refers to antibody binding to apredetermined antigen. Typically, the antibody binds with an affinity(K_(D)) of approximately less than 10⁻⁷ M, such as approximately lessthan 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower when determined by surfaceplasmon resonance (SPR) technology in a BIACORE 3000 instrument usingrecombinant human IL-15 as the analyte and the antibody as the ligand,and binds to the predetermined antigen with an affinity that is at leasttwo-fold greater than its affinity for binding to a non-specific antigen(e.g., BSA, casein) other than the predetermined antigen or aclosely-related antigen. The phrases “an antibody recognizing anantigen” and “an antibody specific for an antigen” are usedinterchangeably herein with the term “an antibody which bindsspecifically to an antigen”.

The term “K_(D)”, as used herein, is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

As used herein, “nonswitched isotype” refers to the isotypic class ofheavy chain that is produced when no isotype switching has taken place;the CH gene encoding the nonswitched isotype is typically the first CHgene immediately downstream from the functionally rearranged VDJ gene.Isotype switching has been classified as classical or non-classicalisotype switching. Classical isotype switching occurs by recombinationevents which involve at least one switch sequence region in thetransgene. Non-classical isotype switching may occur by, for example,homologous recombination between human σ_(μ) and human Σ_(μ)(δ-associated deletion). Alternative non-classical switching mechanisms,such as intertransgene and/or interchromosomal recombination, amongothers, may occur and effectuate isotype switching.

As used herein, the term “switch sequence” refers to those DNA sequencesresponsible for switch recombination. A “switch donor” sequence,typically a μ switch region, will be 5′ (i.e., upstream) of theconstruct region to be deleted during the switch recombination. The“switch acceptor” region will be between the construct region to bedeleted and the replacement constant region (e.g., γ, ε, etc.). As thereis no specific site where recombination always occurs, the final genesequence will typically not be predictable from the construct.

As used herein, “glycosylation pattern” is defined as the pattern ofcarbohydrate units that are covalently attached to a protein, morespecifically to an immunoglobulin protein. A glycosylation pattern of aheterologous antibody can be characterized as being substantiallysimilar to glycosylation patterns which occur naturally on antibodiesproduced by the species of the nonhuman transgenic animal, when one ofordinary skill in the art would recognize the glycosylation pattern ofthe heterologous antibody as being more similar to said pattern ofglycosylation in the species of the nonhuman transgenic animal than tothe species from which the CH genes of the transgene were derived.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete V_(H) or V_(L) domain, respectively. Arearranged immunoglobulin gene locus can be identified by comparison togermline DNA; a rearranged locus will have at least one recombinedheptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule”, as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., V_(H),V_(L), CDR3) that bind to IL-15, is intended to refer to a nucleic acidmolecule in which the nucleotide sequences encoding the antibody orantibody portion are free of other nucleotide sequences encodingantibodies or antibody portions that bind antigens other than IL-15,which other sequences may naturally flank the nucleic acid in humangenomic DNA. SEQ ID NOS: 1-4 correspond to the nucleotide and amino acidsequences comprising the heavy chain (V_(H)) and light chain (V_(L))variable regions of the human anti-IL-15 antibody 146B7 of theinvention. In particular, SEQ ID NO:1 and 2 correspond to the V_(H) ofthe 146B7 antibody, SEQ ID NO:3 and 4 correspond to the V_(L) of the146B7 antibody.

The present invention also encompasses “conservative sequencemodifications” of the sequences set forth in SEQ ID NOs: 1-4, i.e.,nucleotide and amino acid sequence modifications which do notsignificantly affect or alter the binding characteristics of theantibody encoded by the nucleotide sequence or containing the amino acidsequence. Such conservative sequence modifications include nucleotideand amino acid substitutions, additions and deletions. Modifications canbe introduced into SEQ ID NOs:1-4 by standard techniques known in theart, such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitutions include ones in which the aminoacid residue is replaced with an amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a human anti-IL-15 antibodyis preferably replaced with another amino acid residue from the sameside chain family.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a anti-IL-15 antibody coding sequence,such as by saturation mutagenesis, and the resulting modified anti-IL-15antibodies can be screened for binding activity.

Accordingly, antibodies encoded by the (heavy and light chain variableregion) nucleotide sequences disclosed herein and/or containing the(heavy and light chain variable region) amino acid sequences disclosedherein (i.e., SEQ ID NOs: 1-4) include substantially similar antibodiesencoded by or containing similar sequences which have beenconservatively modified. Further discussion as to how such substantiallysimilar antibodies can be generated based on the partial (i.e., heavyand light chain variable regions) sequences disclosed herein as SEQ IDNos:1-4 is provided below.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

For amino acid sequences, the term “homology” indicates the degree ofidentity between two amino acid sequences when optimally aligned andcompared with appropriate insertions or deletions.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures thereof may be mutated, inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions of the presentinvention can be used to treat a subject with an inflammatory disease,such as arthritis, e.g., rheumatoid arthritis. The term “non-humananimal” includes all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, dog, cow, chickens, amphibians, reptiles,etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Production of Human Antibodies to IL-15

Human monoclonal antibodies of the invention can be produced using avariety of known techniques, such as the standard somatic cellhybridization technique described by Kohler and Milstein, Nature 256:495 (1975). Although somatic cell hybridization procedures arepreferred, in principle, other techniques for producing monoclonalantibodies also can be employed, e.g., viral or oncogenic transformationof B lymphocytes, phage display technique using libraries of humanantibody genes.

The preferred animal system for generating hybridomas which producehuman monoclonal antibodies of the invention is the murine system.Hybridoma production in the mouse is well known in the art, includingimmunization protocols and techniques for isolating and fusing immunizedsplenocytes.

In one embodiment, human monoclonal antibodies directed against IL-15are generated using transgenic or transchromosomal mice carrying partsof the human immune system rather than the mouse system. In oneembodiment, the invention employs transgenic mice, referred to herein as“HuMAb mice” which contain a human immunoglobulin gene miniloci thatencodes unrearranged human heavy (μ and γ) and κ light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg, N. et al. (1994)Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or K, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed inLonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101;Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93,and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci.764:536-546). The preparation of HuMAb mice is described in detail inSection II below and in Taylor, L. et al. (1992) Nucleic Acids Research20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724;Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993)EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920;Lonberg et al., (1994) Nature 368(6474): 856-859; Lonberg, N. (1994)Handbook of Experimental Pharmacology 113:49-101; Taylor, L. et al.(1994) International Immunology 6: 579-591; Lonberg, N. and Huszar, D.(1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N.(1995) Ann. N.Y. Acad. Sci. 764:536-546; Fishwild, D. et al. (1996)Nature Biotechnology 14: 845-851. See further, U.S. Pat. Nos. 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016;5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay, andGenPharm International; U.S. Pat. No. 5,545,807 to Surani et al.;International Publication Nos. WO 98/24884, published on Jun. 11, 1998;WO 94/25585, published Nov. 10, 1994; WO 93/1227, published Jun. 24,1993; WO 92/22645, published Dec. 23, 1992; WO 92/03918, published Mar.19, 1992. The preparation of HCO12 transgenic HuMAb mice, in particular,is described in Example 2.

Immunizations

To generate fully human monoclonal antibodies to IL-15, transgenic ortranschromosomal mice containing human immunoglobulin genes (e.g.,HCo12, HCo7 or KM mice) can be immunized with a purified or enrichedpreparation of the IL-15 antigen and/or cells expressing IL-15, asdescribed, for example, by Lonberg et al. (1994) Nature 368(6474):856-859; Fishwild et al. (1996) Nature Biotechnology 14: 845-851 and WO98/24884. Alternatively, mice can be immunized with DNA encoding humanIL-15. Preferably, the mice will be 6-16 weeks of age upon the firstinfusion. For example, a purified or enriched preparation (5-50 μg) ofthe IL-15 antigen can be used to immunize the HuMAb miceintraperitoneally. In the event that immunizations using a purified orenriched preparation of the IL-15 antigen do not result in antibodies,mice can also be immunized with cells expressing IL-15, e.g., a cellline, to promote immune responses.

Cumulative experience with various antigens has shown that the HuMAbtransgenic mice respond best when initially immunized intraperitoneally(IP) or subcutaneously (SC) with antigen in complete Freund's adjuvant,followed by every other week IP/SC immunizations (up to a total of 10)with antigen in incomplete Freund's adjuvant. The immune response can bemonitored over the course of the immunization protocol with plasmasamples being obtained by retroorbital bleeds. The plasma can bescreened by ELISA (as described below), and mice with sufficient titersof anti-IL-15 human immunoglobulin can be used for fusions. Mice can beboosted intravenously with antigen 3 days before sacrifice and removalof the spleen.

Generation of Hybridomas Producing Human Monoclonal Antibodies to IL-15

To generate hybridomas producing human monoclonal antibodies to IL-15,splenocytes and lymph node cells from immunized mice can be isolated andfused to an appropriate immortalized cell line, such as a mouse myelomacell line. The resulting hybridomas can then be screened for theproduction of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toSP2/0-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50%PEG (w/v). Cells can be plated at approximately 1×10⁵ in flat bottommicrotiter plate, followed by a two week incubation in selective mediumcontaining besides usual reagents 10% fetal Clone Serum, 5-10% origenhybridoma cloning factor (IGEN) and 1×HAT (Sigma). After approximatelytwo weeks, cells can be cultured in medium in which the HAT is replacedwith HT. Individual wells can then be screened by ELISA for humananti-IL-15 monoclonal IgM and IgG antibodies. Once extensive hybridomagrowth occurs, medium can be observed usually after 10-14 days. Theantibody secreting hybridomas can be replated, screened again, and ifstill positive for human IgG, anti-IL-15 monoclonal antibodies can besubcloned at least twice by limiting dilution. The stable subclones canthen be cultured in vitro to generate antibody in tissue culture mediumfor characterization.

Generation of Transfectomas Producing Human Monoclonal Antibodies toIL-15

Human antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(Morrison, S. (1985) Science 229:1202).

For example, in one embodiment, the gene(s) of interest, e.g., humanantibody genes, can be ligated into an expression vector such as aeukaryotic expression plasmid such as used by GS gene expression systemdisclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expressionsystems well known in the art. The purified plasmid with the clonedantibody genes can be introduced in eukaryotic host cells such asCHO-cells or NSO-cells or alternatively other eukaryotic cells like aplant derived cells, fungi or yeast cells. The method used to introducethese genes could be methods described in the art such aselectroporation, lipofectine, lipofectamine or other. After introducingthese antibody genes in the host cells, cells expressing the antibodycan be identified and selected. These cells represent the transfectomaswhich can then be amplified for their expression level and upscaled toproduce antibodies. Recombinant antibodies can be isolated and purifiedfrom these culture supernatants and/or cells.

Alternatively these cloned antibody genes can be expressed in otherexpression systems such as E. coli or in complete organisms or can besynthetically expressed.

Use of Partial Antibody Sequences to Express Intact Antibodies

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998, Nature332:323-327; Jones, P. et al., 1986, Nature 321:522-525; and Queen, C.et al., 1989, Proc. Natl. Acad. See. U.S.A. 86:10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline antibody gene sequences. These germline sequences willdiffer from mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V(D)J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody atindividual evenly across the variable region. For example, somaticmutations are relatively infrequent in the amino-terminal portion offramework region. For example, somatic mutations are relativelyinfrequent in the amino terminal portion of framework region 1 and inthe carboxy-terminal portion of framework region 4. Furthermore, manysomatic mutations do not significantly alter the binding properties ofthe antibody. For this reason, it is not necessary to obtain the entireDNA sequence of a particular antibody in order to recreate an intactrecombinant antibody having binding properties similar to those of theoriginal antibody (see PCT/US99/05535 filed on Mar. 12, 1999). Partialheavy and light chain sequence spanning the CDR regions is typicallysufficient for this purpose. The partial sequence is used to determinewhich germline variable and joining gene segments contributed to therecombined antibody variable genes. The germline sequence is then usedto fill in missing portions of the variable regions. Heavy and lightchain leader sequences are cleaved during protein maturation and do notcontribute to the properties of the final antibody. To add missingsequences, cloned cDNA sequences can be combined with syntheticoligonucleotides by ligation or PCR amplification. Alternatively, theentire variable region can be synthesized as a set of short,overlapping, oligonucleotides and combined by PCR amplification tocreate an entirely synthetic variable region clone. This process hascertain advantages such as elimination or inclusion or particularrestriction sites, or optimization of particular codons.

The nucleotide sequences of heavy and light chain transcripts from ahybridoma are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak,1991, J. Biol. Chem. 266L19867019870); and, HindIII sites are engineeredupstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding, and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotide approximately the midpoint of the correspondingnon-coding oligonucleotide. Thus, for each chain, the oligonucleotidescan be assembled into overlapping double stranded sets that spansegments of 150-400 nucleotides. The pools are then used as templates toproduce PCR amplification products of 150-400 nucleotides. Typically, asingle variable region oligonucleotide set will be broken down into twopools which are separately amplified to generate two overlapping PCRproducts. These overlapping products are then combined by PCRamplification to form the complete variable region. It may also bedesirable to include an overlapping fragment of the heavy or light chainconstant region (including the BbsI site of the kappa light chain, orthe AgeI site if the gamma heavy chain) in the PCR amplification togenerate fragments that can easily be cloned into the expression vectorconstructs.

The reconstructed heavy and light chain variable regions are thencombined with cloned promoter, leader sequence, translation initiation,leader sequence, constant region, 3′ untranslated, polyadenylation, andtranscription termination, sequences to form expression vectorconstructs. The heavy and light chain expression constructs can becombined into a single vector, co-transfected, serially transfected, orseparately transfected into host cells which are then fused to form ahost cell expressing both chains.

Plasmids for use in construction of expression vectors for human IgGκare described below (Example 1). The plasmids were constructed so thatPCR amplified V heavy and V kappa light chain cDNA sequences could beused to reconstruct complete heavy and light chain minigenes. Theseplasmids can be used to express completely human IgG₁κ or IgG₄κantibodies. Fully human and chimeric antibodies of the present inventionalso include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. Similarplasmids can be constructed for expression of other heavy chainisotypes, or for expression of antibodies comprising lambda lightchains.

Thus, in another aspect of the invention, the structural features of anhuman anti-IL-15 antibodies of the invention, 146B7, 147H5, 404A8 and404E4, are used to create structurally related human anti-IL-15antibodies that retain at least one functional property of theantibodies of the invention, such as binding to IL-15. Morespecifically, one or more CDR regions of 146B7, 147H5, 404A8 and 404E4can be combined recombinantly with known human framework regions andCDRs to create additional, recombinantly-engineered, human anti-IL-15antibodies of the invention.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-IL-15 antibody comprising:

preparing an antibody comprising (1) human heavy chain framework regionsand human heavy chain CDRs, wherein at least one of the human heavychain CDRs comprises an amino acid sequence selected from the amino acidsequences of CDRs shown in FIG. 2 (or corresponding amino acid residuesin SEQ ID NO: 2); and (2) human light chain framework regions and humanlight chain CDRs, wherein at least one of the human heavy chain CDRscomprises an amino acid sequence selected from the amino acid sequencesof CDRs shown in FIG. 3 (or corresponding amino acid residues in SEQ IDNO: 4);

wherein the antibody retains the ability to bind to IL-15. The abilityof the antibody to bind IL-15 can be determined using standard bindingassays, such as those set forth in the Examples (e.g., an ELISA).

Since it is well known in the art that antibody heavy and light chainCDR3 domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen, the recombinantantibodies of the invention prepared as set forth above preferablycomprise the heavy and light chain CDR3s of 146B7, 147H5, 404A8 and404E4. The antibodies further can comprise the CDR2s of 146B7, 147H5,404A8 and 404E4. The antibodies further can comprise the CDR1s 146B7,147H5, 404A8 and 404E4. The antibodies can further comprise anycombinations of the CDRs.

Accordingly, in another embodiment, the invention further providesanti-IL-15 antibodies comprising: (1) human heavy chain frameworkregions, a human heavy chain CDR1 region, a human heavy chain CDR2region, and a human heavy chain CDR3 region, wherein the human heavychain CDR3 region is selected from the CDR3s of 146B7, 147H5, 404A8 and404E4, for example, a human heavy chain CDR region of 146B7 as shown inFIG. 2 (or corresponding amino acid residues in SEQ ID NO: 2); and (2)human light chain framework regions, a human light chain CDR1 region, ahuman light chain CDR2 region, and a human light chain CDR3 region,wherein the human light chain CDR3 region is selected from the CDR3s of146B7, 147H5, 404A8 and 404E4, for example, a human light chain CDRregion of 146B7 as shown in FIG. 3 (or corresponding amino acid residuesin SEQ ID NO: 4), wherein the antibody binds IL-15. The antibody mayfurther comprise the heavy chain CDR2 and/or the light chain CDR2 of146B7, 147H5, 404A8 and 404E4. The antibody may further comprise theheavy chain CDR1 and/or the light chain CDR1 of 146B7, 147H5, 404A8 and404E4.

The CDR1, 2, and/or 3 regions of the engineered antibodies describedabove can comprise the exact amino acid sequence(s) as those of 146B7,147H5, 404A8 and 404E4 disclosed herein. However, the ordinarily skilledartisan will appreciate that some deviation from the exact CDR sequencesof 146B7, 147H5, 404A8 and 404E4 may be possible while still retainingthe ability of the antibody to bind IL-15 effectively (e.g.,conservative sequence modifications). Accordingly, in anotherembodiment, the engineered antibody may be composed of one or more CDRsthat are, for example, 90%, 95%, 98% or 99.5% identical to one or moreCDRs of 146B7, 147H5, 404A8 and 404E4.

In addition to simply binding IL-15, engineered antibodies such as thosedescribed above may be selected for their retention of other functionalproperties of antibodies of the invention, such as:

(1) binding to human IL-15 and inhibiting IL-15 induced proinflammatoryeffects;

(2) inhibiting IL-15 induced TNFα production or T cell proliferation;

(3) binding to human IL-15 with a dissociation equilibrium constant(K_(D)) of less than approximately 10⁻⁷ M when determined by surfaceplasmon resonance

(SPR) technology in a BIACORE 3000 instrument using recombinant humanIL-15 as the analyte and the antibody as the ligand;

(4) binding to an epitope located on the β- and/or γ-chain interactingdomain of human IL-15;

(5) interfering with the binding of Asp⁸ of human IL-15 to the β-unit ofthe human IL-15 receptor and/or of Gln¹⁰⁸ of human IL-15 to the γ-unitof human IL-15 receptor;

(6) binding to receptor-bound human IL-15;

(7) binding to human IL-15 and inhibiting the ability of human IL-15 toinduce parakeratosis;

(8) binding to human IL-15 and inhibiting the ability of human IL-15 toinduce epidermal thickening;

(9) binding to human IL-15 and inhibiting the ability of human IL-15 toinduce proliferation of keratinocytes; and/or

(10) binding to human IL-15 and inhibiting the ability of human IL-15 toinduce chemotaxis of activated leukocytes.

Characterization of Human Monoclonal Antibodies to IL-15

Human monoclonal antibodies of the invention can be characterized forbinding to IL-15 using a variety of known techniques. Generally, theantibodies are initially characterized by ELISA. Briefly, microtiterplates can be coated with purified IL-15 in PBS, and then blocked withirrelevant proteins such as bovine serum albumin (BSA) diluted in PBS.Dilutions of plasma from IL-15-immunized mice are added to each well andincubated for 1-2 hours at 37° C. The plates are washed with PBS/Tween20 and then incubated with a goat-anti-human IgG Fc-specific polyclonalreagent conjugated to alkaline phosphatase for 1 hour at 37° C. Afterwashing, the plates are developed with ABTS substrate, and analyzed atOD of 405. Preferably, mice which develop the highest titers will beused for fusions.

An ELISA assay as described above can be used to screen for antibodiesand, thus, hybridomas that produce antibodies that show positivereactivity with the IL-15 immunogen. Hybridomas that bind, preferablywith high affinity, to IL-15 can than be subcloned and furthercharacterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can then be chosen for makinga cell bank, and for antibody purification.

To purify human anti-IL-15 antibodies, selected hybridomas can be grownin roller bottles, two-liter spinner-flasks or other culture systems.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.) topurify the protein. After buffer exchange to PBS, the concentration canbe determined by OD₂₈₀ using 1.43 extinction coefficient or preferablyby nephelometric analysis. IgG can be checked by gel electrophoresis andby antigen specific method.

To determine if the selected human anti-IL-15 monoclonal antibodies bindto unique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Biotinylated MAb bindingcan be detected with a streptavidin labeled probe. To determine theisotype of purified antibodies, isotype ELISAs can be performed usingart recognized techniques. For example, wells of microtiter plates canbe coated with 10 μg/ml of anti-human Ig overnight at 4° C. Afterblocking with 5% BSA, the plates are reacted with 10 μg/ml of monoclonalantibodies or purified isotype controls, at ambient temperature for twohours. The wells can then be reacted with either human IgG1 or otherhuman isotype specific conjugated probes. Plates are developed andanalyzed as described above.

To test the binding of monoclonal antibodies to live cells expressingIL-15, flow cytometry can be used. Briefly, cell lines and/or humanPBMCs expressing membrane-bound IL-15 (grown under standard growthconditions) are mixed with various concentrations of monoclonalantibodies in PBS containing 0.1% BSA and 0.01% NaN3 at 4° C. for 1hour. After washing, the cells are reacted with Fluorescein-labeledanti-human IgG antibody under the same conditions as the primaryantibody staining. The samples can be analyzed by FACScan instrumentusing light and side scatter properties to gate on single cells andbinding of the labeled antibodies is determined. An alternative assayusing fluorescence microscopy may be used (in addition to or instead of)the flow cytometry assay. Cells can be stained exactly as describedabove and examined by fluorescence microscopy. This method allowsvisualization of individual cells, but may have diminished sensitivitydepending on the density of the antigen.

Anti-IL-15 human IgGs can be further tested for reactivity with theIL-15 antigen by Western blotting. Briefly, cell extracts from cellsexpressing IL-15 can be prepared and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. After electrophoresis, the separatedantigens will be transferred to nitrocellulose membranes, blocked with20% mouse serum, and probed with the monoclonal antibodies to be tested.Human IgG binding can be detected using anti-human IgG alkalinephosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem.Co., St. Louis, Mo.).

II. Production of Transgenic and Transchromosomal Nonhuman Animals whichGenerate Human Monoclonal Anti-IL-15 Antibodies

In yet another aspect, the invention provides transgenic andtranschromosomal non-human animals, such as transgenic ortranschromosomal mice, which are capable of expressing human monoclonalantibodies that specifically bind to IL-15. In a particular embodiment,the invention provides a transgenic or transchromosomal mouse having agenome comprising a human heavy chain transgene, such that the mouseproduces human anti-IL-15 antibodies when immunized with IL-15 antigenand/or cells expressing IL-15. The human heavy chain transgene can beintegrated into the chromosomal DNA of the mouse, as is the case fortransgenic, e.g., HuMAb mice, as described in detail herein andexemplified. Alternatively, the human heavy chain transgene can bemaintained extrachromosomally, as is the case for transchromosomal(e.g., KM) mice as described in WO 02/43478. Such transgenic andtranschromosomal mice are capable of producing multiple isotypes ofhuman monoclonal antibodies to IL-15 (e.g., IgG, IgA and/or IgE) byundergoing V-D-J recombination and isotype switching. Isotype switchingmay occur by, e.g., classical or non-classical isotype switching.

The design of a transgenic or transchromsomal non-human animal thatresponds to foreign antigen stimulation with a heterologous antibodyrepertoire, requires that the heterologous immunoglobulin transgenescontained within the transgenic animal function correctly throughout thepathway of B-cell development. This includes, for example, isotypeswitching of the heterologous heavy chain transgene. Accordingly,transgenes are constructed so as to produce isotype switching and one ormore of the following of antibodies: (1) high level and cell-typespecific expression, (2) functional gene rearrangement, (3) activationof and response to allelic exclusion, (4) expression of a sufficientprimary repertoire, (5) signal transduction, (6) somatic hypermutation,and (7) domination of the transgene antibody locus during the immuneresponse.

Not all of the foregoing criteria need be met. For example, in thoseembodiments wherein the endogenous immunoglobulin loci of the transgenicanimal are functionally disrupted, the transgene need not activateallelic exclusion. Further, in those embodiments wherein the transgenecomprises a functionally rearranged heavy and/or light chainimmunoglobulin gene, the second criteria of functional generearrangement is unnecessary, at least for that transgene which isalready rearranged. For background on molecular immunology, see,Fundamental Immunology, 2nd edition (1989), Paul William E., ed. RavenPress, N.Y.

In certain embodiments, the transgenic or transchromosomal non-humananimals used to generate the human monoclonal antibodies of theinvention contain rearranged, unrearranged or a combination ofrearranged and unrearranged heterologous immunoglobulin heavy and lightchain transgenes in the germline of the transgenic animal. Each of theheavy chain transgenes comprises at least one C_(H) gene. In addition,the heavy chain transgene may contain functional isotype switchsequences, which are capable of supporting isotype switching of aheterologous transgene encoding multiple C_(H) genes in the B-cells ofthe transgenic animal. Such switch sequences may be those which occurnaturally in the germline immunoglobulin locus from the species thatserves as the source of the transgene C_(H) genes, or such switchsequences may be derived from those which occur in the species that isto receive the transgene construct (the transgenic animal). For example,a human transgene construct that is used to produce a transgenic mousemay produce a higher frequency of isotype switching events if itincorporates switch sequences similar to those that occur naturally inthe mouse heavy chain locus, as presumably the mouse switch sequencesare optimized to function with the mouse switch recombinase enzymesystem, whereas the human switch sequences are not. Switch sequences maybe isolated and cloned by conventional cloning methods, or may besynthesized de novo from overlapping synthetic oligonucleotides designedon the basis of published sequence information relating toimmunoglobulin switch region sequences (Mills et al., Nucl. Acids Res.15:7305-7316 (1991); Sideras et al., Intl. Immunol. 1:631-642 (1989)).For each of the foregoing transgenic animals, functionally rearrangedheterologous heavy and light chain immunoglobulin transgenes are foundin a significant fraction of the B-cells of the transgenic animal (atleast 10 percent).

The transgenes used to generate the transgenic animals of the inventioninclude a heavy chain transgene comprising DNA encoding at least onevariable gene segment, one diversity gene segment, one joining genesegment and at least one constant region gene segment. Theimmunoglobulin light chain transgene comprises DNA encoding at least onevariable gene segment, one joining gene segment and at least oneconstant region gene segment. The gene segments encoding the light andheavy chain gene segments are heterologous to the transgenic non-humananimal in that they are derived from, or correspond to, DNA encodingimmunoglobulin heavy and light chain gene segments from a species notconsisting of the transgenic non-human animal. In one aspect of theinvention, the transgene is constructed such that the individual genesegments are unrearranged, i.e., not rearranged so as to encode afunctional immunoglobulin light or heavy chain. Such unrearrangedtransgenes support recombination of the V, D, and J gene segments(functional rearrangement) and preferably support incorporation of allor a portion of a D region gene segment in the resultant rearrangedimmunoglobulin heavy chain within the transgenic non-human animal whenexposed to the IL-15 antigen.

In an alternate embodiment, the transgenes comprise an unrearranged“mini-locus”. Such transgenes typically comprise a substantial portionof the C, D, and J segments as well as a subset of the V gene segments.In such transgene constructs, the various regulatory sequences, e.g.promoters, enhancers, class switch regions, splice-donor andsplice-acceptor sequences for RNA processing, recombination signals andthe like, comprise corresponding sequences derived from the heterologousDNA. Such regulatory sequences may be incorporated into the transgenefrom the same or a related species of the non-human animal used in theinvention. For example, human immunoglobulin gene segments may becombined in a transgene with a rodent immunoglobulin enhancer sequencefor use in a transgenic mouse. Alternatively, synthetic regulatorysequences may be incorporated into the transgene, wherein such syntheticregulatory sequences are not homologous to a functional DNA sequencethat is known to occur naturally in the genomes of mammals. Syntheticregulatory sequences are designed according to consensus rules, such as,for example, those specifying the permissible sequences of asplice-acceptor site or a promoter/enhancer motif. For example, aminilocus comprises a portion of the genomic immunoglobulin locus havingat least one internal (i.e., not at a terminus of the portion) deletionof a non-essential DNA portion (e.g., intervening sequence; intron orportion thereof) as compared to the naturally-occurring germline Iglocus.

In a preferred embodiment of the invention, the transgenic ortranschromosomal animal used to generate human antibodies to IL-15contains at least one, typically 2-10, and sometimes 25-50 or morecopies of the transgene described in Example 12 of WO 98/24884 (e.g.,pHC1 or pHC2) bred with an animal containing a single copy of a lightchain transgene described in Examples 5, 6, 8, or 14 of WO 98/24884, andthe offspring bred with the J_(H) deleted animal described in Example 10of WO 98/24884. Animals are bred to homozygosity for each of these threetraits. Such animals have the following genotype: a single copy (perhaploid set of chromosomes) of a human heavy chain unrearrangedmini-locus (described in Example 12 of WO 98/24884), a single copy (perhaploid set of chromosomes) of a rearranged human K light chainconstruct (described in Example 14 of WO 98/24884), and a deletion ateach endogenous mouse heavy chain locus that removes all of thefunctional J_(H) segments (described in Example 10 of WO 98/24884). Suchanimals are bred with mice that are homozygous for the deletion of theJ_(H) segments (Examples 10 of WO 98/24884) to produce offspring thatare homozygous for the J_(H) deletion and hemizygous for the human heavyand light chain constructs. The resultant animals are injected withantigens and used for production of human monoclonal antibodies againstthese antigens.

B cells isolated from such an animal are monospecific with regard to thehuman heavy and light chains because they contain only a single copy ofeach gene. Furthermore, they will be monospecific with regards to humanor mouse heavy chains because both endogenous mouse heavy chain genecopies are nonfunctional by virtue of the deletion spanning the J_(H)region introduced as described in Example 9 and 12 of WO 98/24884.Furthermore, a substantial fraction of the B cells will be monospecificwith regards to the human or mouse light chains because expression ofthe single copy of the rearranged human κ light chain gene willallelically and isotypically exclude the rearrangement of the endogenousmouse K and lambda chain genes in a significant fraction of B-cells.

Transgenic and transchromsomal mice employed in the present inventionexhibit immunoglobulin production with a significant repertoire, ideallysubstantially similar to that of a native mouse. Thus, for example, inembodiments where the endogenous Ig genes have been inactivated, thetotal immunoglobulin levels will range from about 0.1 to 10 mg/ml ofserum, preferably 0.5 to 5 mg/ml, ideally at least about 1.0 mg/ml. Whena transgene capable of effecting a switch to IgG from IgM has beenintroduced into the transgenic mouse, the adult mouse ratio of serum IgGto IgM is preferably about 10:1. The IgG to IgM ratio will be much lowerin the immature mouse. In general, greater than about 10%, preferably 40to 80% of the spleen and lymph node B cells express exclusively humanIgG protein.

The repertoire will ideally approximate that shown in a native mouse,usually at least about 10% as high, preferably 25 to 50% or more.Generally, at least about a thousand different immunoglobulins (ideallyIgG), preferably 10⁴ to 10⁶ or more, will be produced, dependingprimarily on the number of different V, J and D regions introduced intothe mouse genome. These immunoglobulins will typically recognize aboutone-half or more of highly antigenic proteins, e.g., staphylococcusprotein A. Typically, the immunoglobulins will exhibit an affinity(K_(D)) for preselected antigens of below 10⁻⁷ M, such as of below 10⁻⁸M, 10⁻⁹ M or 10⁻¹⁰ M or even lower.

In some embodiments, it may be preferable to generate mice withpredetermined repertoires to limit the selection of V genes representedin the antibody response to a predetermined antigen type. A heavy chaintransgene having a predetermined repertoire may comprise, for example,human V_(H) genes which are preferentially used in antibody responses tothe predetermined antigen type in humans. Alternatively, some V_(H)genes may be excluded from a defined repertoire for various reasons(e.g., have a low likelihood of encoding high affinity V regions for thepredetermined antigen; have a low propensity to undergo somatic mutationand affinity sharpening; or are immunogenic to certain humans). Thus,prior to rearrangement of a transgene containing various heavy or lightchain gene segments, such gene segments may be readily identified, e.g.by hybridization or DNA sequencing, as being from a species of organismother than the transgenic animal.

Transgenic and transchromosomal mice as described above can be immunizedwith, for example, a purified or enriched preparation of IL-15 antigenand/or cells expressing IL-15. Alternatively, the transgenic mice can beimmunized with DNA encoding human IL-15. The mice will then produce Bcells which undergo class-switching via intratransgene switchrecombination (cis-switching) and express immunoglobulins reactive withIL-15. The immunoglobulins can be human antibodies (also referred to as“human sequence antibodies”), wherein the heavy and light chainpolypeptides are encoded by human transgene sequences, which may includesequences derived by somatic mutation and V region recombinatorialjoints, as well as germline-encoded sequences; these human antibodiescan be referred to as being substantially identical to a polypeptidesequence encoded by a human V_(L) or V_(H) gene segment and a humanJ_(L) or D_(H) and J_(H) segment, even though other non-germlinesequences may be present as a result of somatic mutation anddifferential V-J and V-D-J recombination joints. The variable regions ofeach antibody chain are typically at least 80 percent encoded by humangermline V, J, and, in the case of heavy chains, D, gene segments;frequently at least 85 percent of the variable regions are encoded byhuman germline sequences present on the transgene; often 90 or 95percent or more of the variable region sequences are encoded by humangermline sequences present on the transgene. However, since non-germlinesequences are introduced by somatic mutation and VJ and VDJ joining, thehuman sequence antibodies will frequently have some variable regionsequences (and less frequently constant region sequences) which are notencoded by human V, D, or J gene segments as found in the humantransgene(s) in the germline of the mice. Typically, such non-germlinesequences (or individual nucleotide positions) will cluster in or nearCDRs, or in regions where somatic mutations are known to cluster.

Human antibodies which bind to the predetermined antigen can result fromisotype switching, such that human antibodies comprising a humansequence γ chain (such as γ1, γ2a, γ2B, or γ3) and a human sequencelight chain (such as kappa) are produced. Such isotype-switched humanantibodies often contain one or more somatic mutation(s), typically inthe variable region and often in or within about 10 residues of a CDR)as a result of affinity maturation and selection of B cells by antigen,particularly subsequent to secondary (or subsequent) antigen challenge.These high affinity human antibodies may have binding affinities (K_(D))of below 10⁻⁷ M, such as of below 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or evenlower.

Another aspect of the invention includes B cells derived from transgenicor transchromosomal mice as described herein. The B cells can be used togenerate hybridomas expressing human monoclonal antibodies which bindwith high affinity (e.g., lower than 10⁻⁷ M) to human IL-15. Thus, inanother embodiment, the invention provides a hybridoma which produces ahuman antibody having an affinity (K_(D)) of below 10⁻⁷ M, such as ofbelow 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower when determined by surfaceplasmon resonance (SPR) technology in a BIACORE 3000 instrument usingrecombinant human IL-15 as the analyte and the antibody as the ligandfor binding human IL-15, wherein the antibody comprises:

a human sequence light chain composed of (1) a light chain variableregion having a polypeptide sequence which is substantially identical toa polypeptide sequence encoded by a human V_(L) gene segment and a humanJ_(L) segment, and (2) a light chain constant region having apolypeptide sequence which is substantially identical to a polypeptidesequence encoded by a human C_(L) gene segment; and

a human sequence heavy chain composed of a (1) a heavy chain variableregion having a polypeptide sequence which is substantially identical toa polypeptide sequence encoded by a human V_(H) gene segment, optionallya D region, and a human J_(H) segment, and (2) a constant region havinga polypeptide sequence which is substantially identical to a polypeptidesequence encoded by a human C_(H) gene segment.

The development of high affinity human monoclonal antibodies againstIL-15 can be facilitated by a method for expanding the repertoire ofhuman variable region gene segments in a transgenic mouse having agenome comprising an integrated human immunoglobulin transgene, saidmethod comprising introducing into the genome a V gene transgenecomprising V region gene segments which are not present in saidintegrated human immunoglobulin transgene. Often, the V region transgeneis a yeast artificial chromosome comprising a portion of a human V_(H)or V_(L) (V_(K)) gene segment array, as may naturally occur in a humangenome or as may be spliced together separately by recombinant methods,which may include out-of-order or omitted V gene segments. Often atleast five or more functional V gene segments are contained on the YAC.In this variation, it is possible to make a transgenic mouse produced bythe V repertoire expansion method, wherein the mouse expresses animmunoglobulin chain comprising a variable region sequence encoded by aV region gene segment present on the V region transgene and a C regionencoded on the human Ig transgene. By means of the V repertoireexpansion method, transgenic mice having at least 5 distinct V genes canbe generated; as can mice containing at least about 24 V genes or more.Some V gene segments may be non-functional (e.g., pseudogenes and thelike); these segments may be retained or may be selectively deleted byrecombinant methods available to the skilled artisan, if desired.

Once the mouse germline has been engineered to contain a functional YAChaving an expanded V segment repertoire, substantially not present inthe human Ig transgene containing the J and C gene segments, the traitcan be propagated and bred into other genetic backgrounds, includingbackgrounds where the functional YAC having an expanded V segmentrepertoire is bred into a mouse germline having a different human Igtransgene. Multiple functional YACs having an expanded V segmentrepertoire may be bred into a germline to work with a human Ig transgene(or multiple human Ig transgenes). Although referred to herein as YACtransgenes, such transgenes when integrated into the genome maysubstantially lack yeast sequences, such as sequences required forautonomous replication in yeast; such sequences may optionally beremoved by genetic engineering (e.g., restriction digestion andpulsed-field gel electrophoresis or other suitable method) afterreplication in yeast is no longer necessary (i.e., prior to introductioninto a mouse ES cell or mouse prozygote). Methods of propagating thetrait of human sequence immunoglobulin expression, include breeding atransgenic mouse having the human Ig transgene(s), and optionally alsohaving a functional YAC having an expanded V segment repertoire. BothV_(H) and V_(L) gene segments may be present on the YAC. The transgenicmouse may be bred into any background desired by the practitioner,including backgrounds harboring other human transgenes, including humanIg transgenes and/or transgenes encoding other human lymphocyteproteins. The invention also provides a high affinity human sequenceimmunoglobulin produced by a transgenic mouse having an expanded Vregion repertoire YAC transgene. Although the foregoing describes apreferred embodiment of the transgenic animal of the invention, otherembodiments are contemplated which have been classified in fourcategories:

I. Transgenic animals containing an unrearranged heavy and rearrangedlight immunoglobulin transgene;

II. Transgenic animals containing an unrearranged heavy and unrearrangedlight immunoglobulin transgene;

III. Transgenic animal containing rearranged heavy and an unrearrangedlight immunoglobulin transgene; and

IV. Transgenic animals containing rearranged heavy and rearranged lightimmunoglobulin transgenes.

Of these categories of transgenic animal, the preferred order ofpreference is as follows II>I>III>IV where the endogenous light chaingenes (or at least the K gene) have been knocked out by homologousrecombination (or other method) and I>II>III>IV where the endogenouslight chain genes have not been knocked out and must be dominated byallelic exclusion.

III. Antibody Conjugates/Immunotoxins

In another aspect, the present invention features a human anti-IL-15monoclonal antibody conjugated to a therapeutic moiety, such as acytotoxin, a drug (e.g., an immunosuppressant) or a radioisotope. Whenconjugated to a cytotoxin, these antibody conjugates are referred to as“immunotoxins.” A cytotoxin or cytotoxic agent includes any agent thatis detrimental to (e.g., kills) cells. Examples include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). An antibody of the presentinvention can be conjugated to a radioisotope, e.g., radioactive iodine,to generate cytotoxic radiopharmaceuticals for treating a IL-15-relateddisorder, such as a cancer.

The antibody conjugates of the invention can be used to modify a givenbiological response. The therapeutic moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other cytokines or growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

IV. Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination of humanmonoclonal antibodies, or antigen-binding portion(s) thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier. In a preferred embodiment, the compositions includea combination of multiple (e.g., two or more) isolated human antibodiesof the invention. Preferably, each of the antibodies of the compositionbinds to a distinct, pre-selected epitope of IL-15.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a composition of the present inventionwith at least one or more additional therapeutic agents, such asanti-inflammatory agents, DMARDs (disease-modifying anti-rheumaticdrugs), immunosuppressive agents, chemotherapeutics, and psoriasisagents. The pharmaceutical compositions of the invention can also beadministered in conjunction with radiation therapy. Co-administrationwith other antibodies, such as CD4 specific antibodies and IL-2 specificantibodies, are also encompassed by the invention. Such combinationswith CD4 specific antibodies or IL-2 specific antibodies are consideredparticularly useful for treating autoimmune diseases and transplantrejections.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifingalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,bispecific and multispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

To administer a compound of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. For example, the humanantibodies of the invention may be administered once or twice weekly bysubcutaneous injection or once or twice monthly by subcutaneousinjection. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present inventioninclude those suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect. Generally, out of one hundredpercent, this amount will range from about 0.001 percent to about ninetypercent of active ingredient, preferably from about 0.005 percent toabout 70 percent, most preferably from about 0.01 percent to about 30percent.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate. Dosage forms for the topical or transdermaladministration of compositions of this invention include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.001 to 90% (morepreferably, 0.005 to 70%, such as 0.01 to 30%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. A physician orveterinarian having ordinary skill in the art can readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician or veterinarian could start dosesof the compounds of the invention employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. In general, a suitable daily dose of acompositions of the invention will be that amount of the compound whichis the lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described above.It is preferred that administration be intravenous, intramuscular,intraperitoneal, or subcutaneous, preferably administered proximal tothe site of the target. If desired, the effective daily dose of atherapeutic compositions may be administered as two, three, four, five,six or more sub-doses administered separately at appropriate intervalsthroughout the day, optionally, in unit dosage forms. While it ispossible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation (composition).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.No. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many othersuch implants, delivery systems, and modules are known to those skilledin the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134),different species of which may comprise the formulations of theinventions, as well as components of the invented molecules; p120(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273. In one embodiment of the invention, thetherapeutic compounds of the invention are formulated in liposomes; in amore preferred embodiment, the liposomes include a targeting moiety. Ina most preferred embodiment, the therapeutic compounds in the liposomesare delivered by bolus injection to a site proximal to the tumor orinfection. The composition must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi.

In a further embodiment, the human monoclonal antibodies of theinvention can be formulated to prevent or reduce the transport acrossthe placenta. This can be done by methods known in the art, e.g., byPEGylation of the antibody or by use of F(ab)2′ fragments. Furtherreferences can be made to “Cunningham-Rundles C, Zhuo Z, Griffith B,Keenan J. (1992) Biological activities of polyethylene-glycolimmunoglobulin conjugates. Resistance to enzymatic degradation. J.Immunol Methods. 152:177-190; and to “Landor M. (1995) Maternal-fetaltransfer of immunoglobulins, Ann Allergy Asthma Immunol 74:279-283. Thisis particularly relevant when the antibodies are used for treating orpreventing recurrent spontaneous abortion.

A “therapeutically effective dosage” for rheumatoid arthritis preferablywill result in an ACR20 Preliminary Definition of Improvement in thepatients, more preferred in an ACR50 Preliminary Definition ofImprovement and even more preferred in an ARCD70 Preliminary Definitionof Improvement.

ACR20 Preliminary Definition of Improvement is defined as: ≧20%improvement in: Tender Joint Count (TCJ) and Swollen Joint Count (SWJ)and ≧20% improvement in 3 of following 5 assessments: Patient PainAssessment (VAS), Patient Global assessment (VAS), Physician GlobalAssessment (VAS), Patent Self-Assessed Disability (HAQ), Acute PhaseReactant (CRP or ESR).

ACR50 and ACR70 are defined in the same way with ≧50% and ≧70%improvements, respectively. For further details see Felson et al. inAmerican College of Rheumatology Preliminary Definition of Improvementin Rheumatoid Arthritis; Arthritis Rheumatism (1995) 38: 727-735.

The ability of a compound to inhibit cancer can be evaluated in ananimal model system predictive of efficacy in human tumors.Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to inhibit, such inhibition invitro by assays known to the skilled practitioner. A therapeuticallyeffective amount of a therapeutic compound can decrease tumor size, orotherwise ameliorate symptoms in a subject. One of ordinary skill in theart would be able to determine such amounts based on such factors as thesubject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected.

The ability of the antibodies to treat or prevent psoriasis can also beevaluated according to methods well known in the art.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound may be orally administered, for example, with an inert diluentor an assimilable edible carrier.

V. Uses and Methods of the Invention

Human anti-IL-15 antibodies to IL-15 of the present invention (includingderivatives and conjugates of the antibodies) and compositionscontaining the antibodies can be used in a variety of in vitro and invivo diagnostic and therapeutic applications.

In one embodiment, human antibodies of the invention are used to inhibitIL-15 induced TNFα production by T cells and/or monocytes/macrophages,preferably without inhibiting TNFα production induced by othercytokines, such as IL-2. By contacting the antibody with IL-15 (e.g., byadministering the antibody to a subject), the ability of IL-15 to signalthrough the IL-15 receptor is inhibited and, thus, the production ofTNFα by T-cells and/or monocytes/macrophages also is inhibited.Preferred antibodies bind to epitopes (e.g., particular subunits, suchas the gamma subunit) which are specific to IL-15 and, thus,advantageously inhibit IL-15-induced TNFα production, but do notinterfere with TNFα production by structurally related cytokines, suchas IL-2.

Alternatively, human antibodies are used to interfere with IL-15receptor α-, β- and γ-chain assembly and/or inhibit assembly onneighbouring cells expressing β- and γ-chains as part of the IL-15receptor or another cytokine receptor.

In another embodiment, human antibodies of the invention are used toinhibit IL-15 induced T cell recruitment and/or proliferation,preferably without inhibiting T cell proliferation induced by otherstructurally related cytokines, such as IL-2. As with TNFα production,by contacting the antibody with IL-15 (e.g., by administering theantibody to a subject), the ability of IL-15 to signal through the IL-15receptor is inhibited and, thus, T cell stimulation by IL-15 isinhibited.

Accordingly, in yet another embodiment, the present invention provides amethod for treating or preventing a disorder mediated by IL-15 (e.g., anautoimmune disease, such as psoriasis, rheumatoid arthritis, orinflammatory bowel disease, or an infectious disease, such as HIV), byadministering to a subject a human antibody of the invention in anamount effective to treat or prevent the disorder. The antibody can beadministered alone or along with another therapeutic agent, such as ananti-inflammatory agent, e.g., a steroidal or nonsteroidal inflammatoryagent, or a cytotoxin which acts in conjunction with or synergisticallywith the antibody to treat or prevent the IL-15 mediated disease.

In a particular embodiment, human antibodies of the present inventionare used to treat or to prevent rheumatoid arthritis (RA). Theantibodies limit the role that IL-15 plays in the progression ofinflammation associated with diseases such as RA. T cells, particularlyCD4+ T-helper cells, are involved in the initiation and maintenance ofinflammatory processes in RA. TNF-α, another cytokine, is also involvedin the inflammatory pathways which ultimately lead to joint destructionand incapacitation of the patient with RA. Local synthesis of IL-15plays a key role both in the activation and recruitment of T cells andin the induction of TNF-α and other inflammatory cytokines. The role ofIL-15 in the progression of RA involves a process whereby IL-15, whichis synthesized by macrophages, induces T cell recruitment. The activatedT cells then: (1) maintain macrophage activation; and (2) induce TNF-αproduction. Stimulated macrophages promote the synthesis of more IL-15and T cell activation, thus, continuing the cycle. In addition to itseffects on TNF-α and macrophages, IL-15 also activates neutrophils andaffects local B cell immunoglobulin secretion, particularly rheumatoidfactor synthesis.

Accordingly, anti-IL-15 antibodies of the invention can be used toprevent or block the foregoing effects of IL-15 which cause RA and,thus, can be used to prevent or treat this disease. For example,anti-IL-15 antibodies of the invention can be used to inhibitinflammation and/or prevent chemotaxis of activated leukocytes involvedin RA.

The human antibodies of the present invention may be used for inhibitionof progression of structural damage in patients with rheumatoidarthritis who have had an inadequate response to methotrexate or forreducing sign and symptoms and delaying structural damage in patientswith moderately to severely active rheumatoid arthritis, including thosewho have not previously failed treatment with a DMARD.

Human antibodies of the present invention also can be used to block orinhibit other effects of IL-15. IL-15 is expressed in various cells andtissues including monocytes and macrophages, fibroblasts, dendriticcells, and keratinocytes. Keratinocytes are major constituents of theepidermis and the epithelial lining of mucosal tissue. Control ofkeratinocyte growth is mediated by a complex network of cytokines andgrowth factors, some of which are produced by keratinocytes themselves.Keratinocyte-derived IL-15 contributes to T cell accumulation,proliferation, and survival in psoriatic plaques. Many diseases areknown wherein the number of keratinocytes is increased which leads toepidermal hyperplasia which is responsible for at least some of therelated disease symptoms. These diseases include chronic diseases suchas psoriasis and atopic dermatitis, as well as conditions like chronichand eczema, contact dermatitis, viral warts (HPV associated), cutaneousT cell lymphoma, impaired wound healing, such as impaired wound healingdue to diabetes. Accordingly, the invention provides methods fortreating or preventing such disorders by administering to patients ahuman anti-IL-15 antibody of the invention in an amount effective totreat or prevent the disorder. For example, anti-IL-15 antibodies of theinvention can be used to block or inhibit parakeratosis in psoriasis,reduce epidermal thickness in psoriasis, and reduce proliferation ofkeratinocytes in psoriasis.

IL-15 also modulates the function of intestinal epithelial cells(Reinecker, et al. (1996) Gastroenterology 111: 1706-13). Specifically,IL-15 can cause modifications on mucosal epithelial cells and onintestinal epithelial cell lines and, therefore, is involved in thepathogenesis of inflammatory bowel disease, e.g., celiac disease. Therole of IL-15 in such diseases is shown by the selectiveover-representation of IL-15+ cells in the small intestine of untreatedpatients with celiac disease (WO 00/02582). Thus, it has been shown thatIL-15 is directly involved in the initiation and maintenance of celiacdisease. Accordingly, in another embodiment, anti-IL-15 human antibodiesof the present invention (i.e., which inhibit the proinflammatoryeffects of IL-15) can be used to treat and/or to prevent celiac diseaseby administering the antibody to a patient in an amount effective totreat or prevent the disorder.

In addition, it has been found by the inventors of the present inventionthat IL-15 also promotes the formation of new blood vessels, a processcalled neovascularization or angiogenesis. Accordingly, yet another usefor the antibodies of the invention includes the prevention or treatmentof diseases involving neovascularization. These diseases include avariety of cancers which rely on or are characterized byneovascularization, in addition to inflammatory diseases.

Human antibodies of the present invention also can be used to block orinhibit the effects of IL-15 associated with infectious diseases, suchas HIV. Accordingly, another use for the antibodies of the inventionincludes the prevention or treatment of infectious diseases, e.g.,HIV-1.

For example, the antibodies can be used in vitro or in vivo to diagnosea variety of diseases mediated by IL-15. Specifically, the antibodiescan be used to detect levels of IL-15, or levels of cells which containIL-15 on their membrane surface or linked to their receptors(receptor-bound human IL-15). The detection of such levels of IL-15 canthen be correlated to certain disease symptoms. Alternatively, theantibodies can be used to inhibit or block IL-15 function which, inturn, can prevent or ameliorate disease symptoms caused by IL-15function.

As previously described, human anti-IL-15 antibodies of the inventioncan be co-administered with one or other more therapeutic agents, e.g.,an immunosuppressive agent or an anti-inflammatory agent to increase theoverall anti-inflammatory effect. The antibody can be linked to theagent (as an immunocomplex) or can be administered separate from theagent. In the latter case (separate administration), the antibody can beadministered before, after or concurrently with the agent. Suitabletherapeutic agents include, among others, anti-inflammatory agents,DMARDs (disease-modifying anti-rheumatic drugs), immunosuppressiveagents, chemotherapeutics, and psoriasis agents. The human antibodiesaccording to the invention can also be administered in conjunction withradiation therapy.

In another embodiment, the human antibodies of the invention can beadministered in combination with other antibodies, such as CD4 specificantibodies and IL-2 specific antibodies. A combination of the presenthuman antibodies with CD4 specific antibodies or IL-2 specificantibodies are considered particularly useful for treating autoimmunediseases and transplant rejections.

Also within the scope of the present invention are kits comprising humananti-IL-15 antibodies of the invention and, optionally, instructions foruse. The kit can further contain one or more additional reagents, suchas an immunosuppressive reagent, or one or more additional humanantibodies of the invention (e.g., a human antibody having acomplementary activity which binds to an epitope in the IL-15 antigendistinct from the first human antibody).

Accordingly, patients treated with antibodies of the invention can beadditionally administered (prior to, simultaneously with, or followingadministration of a human antibody of the invention) with anothertherapeutic agent, such as an anti-inflammatory agent, which enhances oraugments the therapeutic effect of the human antibodies.

In yet another embodiment, human antibodies of the invention can be usedto target compounds (e.g., therapeutic agents, labels, cytotoxins,immunosuppressants etc.) to cells which have IL-15 bound to theirsurface (e.g., membrane bound or bound to IL-15 receptor by linking suchcompounds to the antibody. Thus, the invention also provides methods forlocalizing ex vivo, in vivo or in vitro cells expressing IL-15 and IL-15receptor (e.g., with a detectable label, such as a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor).

Other embodiments of the present invention are described in thefollowing Examples.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents ofSequence Listing, figures and all references, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

EXAMPLES Example 1 Generation of Cmu Targeted Mice Construction of a CMDTargeting Vector

The plasmid pICEmu contains an EcoRI/XhoI fragment of the murine Igheavy chain locus, spanning the mu gene, that was obtained from a Balb/Cgenomic lambda phage library (Marcu et al. Cell 22: 187, 1980). Thisgenomic fragment was subcloned into the XhoI/EcoRI sites of the plasmidpICEMI9H (Marsh et al; Gene 32, 481-485, 1984). The heavy chainsequences included in pICEmu extend downstream of the EcoRI site locatedjust 3′ of the mu intronic enhancer, to the XhoI site locatedapproximately 1 kb downstream of the last transmembrane exon of the mugene; however, much of the mu switch repeat region has been deleted bypassage in E. coli.

The targeting vector was constructed as follows. A 1.3 kb HindIII/SmaIfragment was excised from pICEmu and subcloned into HindIII/SmaIdigested pBluescript (Stratagene, La Jolla, Calif.). This pICEmufragment extends from the HindIII site located approximately 1 kb 5′ ofCmu1 to the SmaI site located within Cmu1. The resulting plasmid wasdigested with SmaI/SpeI and the approximately 4 kb SmaI/XbaI fragmentfrom pICEmu, extending from the Sma I site in Cmu1 3′ to the XbaI sitelocated just downstream of the last Cmu exon, was inserted. Theresulting plasmid, pTAR1, was linearized at the SmaI site, and a neoexpression cassette inserted. This cassette consists of the neo geneunder the transcriptional control of the mouse phosphoglycerate kinase(pgk) promoter (XbaI/TaqI fragment; Adra et al. (1987) Gene 60: 65-74)and containing the pgk polyadenylation site (PvuII/HindIII fragment;Boer et al. (1990) Biochemical Genetics 28: 299-308). This cassette wasobtained from the plasmid pKJ1 (described by Tybulewicz et al. (1991)Cell 65: 1153-1163) from which the neo cassette was excised as anEcoRI/HindIII fragment and subcloned into EcoRI/HindIII digestedpGEM-7Zf (+) to generate pGEM-7 (KJ1). The neo cassette was excised frompGEM-7 (KJ1) by EcoRI/SalI digestion, blunt ended and subcloned into theSmaI site of the plasmid pTAR1, in the opposite orientation of thegenomic Cmu sequences. The resulting plasmid was linearized with Not I,and a herpes simplex virus thymidine kinase (tk) cassette was insertedto allow for enrichment of ES clones bearing homologous recombinants, asdescribed by Mansour et al. (1988) Nature 336: 348-352. This cassetteconsists of the coding sequences of the tk gene bracketed by the mousepgk promoter and polyadenylation site, as described by Tybulewicz et al.(1991) Cell 65: 1153-1163. The resulting CMD targeting vector contains atotal of approximately 5.3 kb of homology to the heavy chain locus andis designed to generate a mutant mu gene into which has been inserted aneo expression cassette in the unique SmaI site of the first Cmu exon.The targeting vector was linearized with PvuI, which cuts within plasmidsequences, prior to electroporation into ES cells.

Generation and Analysis of Targeted ES Cells

AB-1 ES cells (McMahon, A. P. and Bradley, A., (1990) Cell 62:1073-1085) were grown on mitotically inactive SNL76/7 cell feeder layers(ibid.) essentially as described (Robertson, E. J. (1987) inTeratocarcinomas and Embryonic Stem Cells: a Practical Approach (E. J.Robertson, ed.) Oxford: IRL Press, p. 71-112). The linearized CMDtargeting vector was electroporated into AB-1 cells by the methodsdescribed Hasty et al. (Hasty, P. R. et al. (1991) Nature 350: 243-246).Electroporated cells were plated into 100 mm dishes at a density of1−2×10⁶ cells/dish. After 24 hours, G418 (200 micrograms/ml of activecomponent) and FIAU (5×10⁻⁷ M) were added to the medium, anddrug-resistant clones were allowed to develop over 8-9 days. Clones werepicked, trypsinized, divided into two portions, and further expanded.Half of the cells derived from each clone were then frozen and the otherhalf analyzed for homologous recombination between vector and targetsequences.

DNA analysis was carried out by Southern blot hybridization. DNA wasisolated from the clones as described by Laird et al. (Laird, P. W. etal., (1991) Nucleic Acids Res. 19: 4293). Isolated genomic DNA wasdigested with SpeI and probed with a 915 bp SacI fragment, probe A (seeFIG. 1), which hybridizes to a sequence between the mu intronic enhancerand the mu switch region. Probe A detects a 9.9 kb SpeI fragment fromthe wild type locus, and a diagnostic 7.6 kb band from a mu locus whichhas homologously recombined with the CMD targeting vector (the neoexpression cassette contains a SpeI site). Of 1132 G418 and FIAUresistant clones screened by Southern blot analysis, 3 displayed the 7.6kb Spe I band indicative of homologous recombination at the mu locus.These 3 clones were further digested with the enzymes BglI, BstXI, andEcoRI to verify that the vector integrated homologously into the mugene. When hybridized with probe A, Southern blots of wild type DNAdigested with BglI, BstXI, or EcoRI produce fragments of 15.7, 7.3, and12.5 kb, respectively, whereas the presence of a targeted mu allele isindicated by fragments of 7.7, 6.6, and 14.3 kb, respectively. All 3positive clones detected by the SpeI digest showed the expected BglI,BstXI, and EcoRI restriction fragments diagnostic of insertion of theneo cassette into the Cmu1 exon.

Generation of Mice Bearing the Mutated Mu Gene

The three targeted ES clones, designated number 264, 272, and 408, werethawed and injected into C57BL/6J blastocysts as described by Bradley(Bradley, A. (1987) in Teratocarcinomas and Embryonic Stem Cells: aPractical Approach. (E. J. Robertson, ed.) Oxford: IRL Press, p.113-151). Injected blastocysts were transferred into the uteri ofpseudopregnant females to generate chimeric mice representing a mixtureof cells derived from the input ES cells and the host blastocyst. Theextent of ES cell contribution to the chimera can be visually estimatedby the amount of agouti coat coloration, derived from the ES cell line,on the black C57BL/6J background. Clones 272 and 408 produced only lowpercentage chimeras (i.e. low percentage of agouti pigmentation) butclone 264 produced high percentage male chimeras. These chimeras werebred with C57BL/6J females and agouti offspring were generated,indicative of germline transmission of the ES cell genome. Screening forthe targeted mu gene was carried out by Southern blot analysis of BglIdigested DNA from tail biopsies (as described above for analysis of EScell DNA). Approximately 50% of the agouti offspring showed ahybridizing BglI band of 7.7 kb in addition to the wild type band of15.7 kb, demonstrating a germline transmission of the targeted mu gene.

Analysis of Transgenic Mice for Functional Inactivation of Mu Gene

To determine whether the insertion of the neo cassette into Cmu1 hasinactivated the Ig heavy chain gene, a clone 264 chimera was bred with amouse homozygous for the JHD mutation, which inactivates heavy chainexpression as a result of deletion of the JH gene segments (Chen et al,(1993) Immunol. 5: 647-656). Four agouti offspring were generated. Serumwas obtained from these animals at the age of 1 month and assayed byELISA for the presence of murine IgM. Two of the four offspring werecompletely lacking IgM (see Table 1). Genotyping of the four animals bySouthern blot analysis of DNA from tail biopsies by BglI digestion andhybridization with probe A (see FIG. 1), and by StuI digestion andhybridization with a 475 bp EcoRI/StuI fragment (ibid.) demonstratedthat the animals which fail to express serum IgM are those in which oneallele of the heavy chain locus carries the JHD mutation, the otherallele the Cmu1 mutation. Mice heterozygous for the JHD mutation displaywild type levels of serum Ig. These data demonstrate that the Cmu1mutation inactivates expression of the mu gene.

TABLE 1 Serum IgM Ig H chain Mouse (micrograms/ml) genotype 42 <0.002CMD/JHD 43 196 +/JHD 44 <0.002 CMD/JHD 45 174 +/JHD 129 × BL6 F1 153 +/+JHD <0.002 JHD/JHDTable 1 shows the levels of serum IgM, detected by ELISA, for micecarrying both the CMD and JHD mutations (CMD/JHD), for mice heterozygousfor the JHD mutation (+/JHD), for wild type (129Sv×C57BL/6J)F1 mice(+/+), and for B cell deficient mice homozygous for the JHD mutation(JHD/JHD).

Example 2 Generation of HCO12 Transgenic Mice The HCO12 Human HeavyChain Transgene

The HCO12 transgene was generated by coinjection of the 80 kb insert ofpHC2 (Taylor et al., 1994, Int. Immunol., 6: 579-591) and the 25 kbinsert of pVx6. The plasmid pVx6 was constructed as described below.

An 8.5 kb HindIII/SalI DNA fragment, comprising the germline human V_(H)1-18 (DP-14) gene together with approximately 2.5 kb of 5′ flanking, and5 kb of 3′ flanking genomic sequence was subcloned into the plasmidvector pSP72 (Promega, Madison, Wis.) to generate the plasmid p343.7.16.A 7 kb BamHI/HindIII DNA fragment, comprising the germline human V_(H)5-51 (DP-73) gene together with approximately 5 kb of 5′ flanking and 1kb of 3′ flanking genomic sequence, was cloned into the pBR322 basedplasmid cloning vector pGP1f (Taylor et al. 1992, Nucleic Acids Res. 20:6287-6295), to generate the plasmid p251f. A new cloning vector derivedfrom pGP1f, pGP1k (SEQ ID NO:13), was digested with EcoRV/BamHI, andligated to a 10 kb EcoRV/BamHI DNA fragment, comprising the germlinehuman V_(H) 3-23 (DP47) gene together with approximately 4 kb of 5′flanking and 5 kb of 3′ flanking genomic sequence. The resultingplasmid, p112.2RR.7, was digested with BamHI/SalI and ligated with the 7kb purified BamHI/SalI insert of p251f. The resulting plasmid, pVx4, wasdigested with XhoI and ligated with the 8.5 kb XhoI/SalI insert ofp343.7.16.

A clone was obtained with the V_(H) 1-18 gene in the same orientation asthe other two V genes. This clone, designated pVx6, was then digestedwith NotI and the purified 26 kb insert coinjected—together with thepurified 80 kb NotI insert of pHC2 at a 1:1 molar ratio—into thepronuclei of one-half day (C57BL/6J×DBA/2J)F2 embryos as described byHogan et al. (B. Hogan et al., Manipulating the Mouse Embryo, ALaboratory Manual, 2^(nd) edition, 1994, Cold Spring Harbor LaboratoryPress, Plainview N.Y.). Three independent lines of transgenic micecomprising sequences from both Vx6 and HC2 were established from micethat developed from the injected embryos. These lines are designated(HCO12)14881, (HCO12)15083, and (HCO12)15087. Each of the three lineswere then bred with mice comprising the CMD mutation described inExample 1, the JKD mutation (Chen et al. 1993, EMBO J. 12: 811-820), andthe (KCo5)9272 transgene (Fishwild et al. 1996, Nature Biotechnology 14:845-851). The resulting mice express human heavy and kappa light chaintransgenes in a background homozygous for disruption of the endogenousmouse heavy and kappa light chain loci.

Example 3 Production of Human Monoclonal Antibodies Against IL-15

HCo12 and HCo7 transgenic mice, generated as described above andsupplied from Medarex, San José, Calif., USA, were immunised with humanrecombinant IL-15 (hIL-15, Immunex corp., Seattle, USA) supplementedwith either Complete Freunds Adjuvant (CFA, lot no. 121024LA, DifcoLaboratories, Detroit, Mich., USA) or with Incomplete Freunds Adjuvant(ICFA, lot no. 121195LA, Difco, subcutaneously (SC) intraperitoneally(IP) or intravenously (IV). In several instances hIL-15 coupled to KLHwas used for immunisation. After several boosts with hIL-15 supplementedwith either Complete or Incomplete Freunds Adjuvant, the serum of themice was tested for the presence of human antibodies directed againstIL-15.

Immunisation Schemes of the Transgenic Mice which Resulted in FinalClones 146B7, 146H5, 404E4 and 404A8

Mouse no. 146 (HCo12), ID 995-146, Female

170699 SC 12 μg hIL-15 in CFA (Difco, Lot no. 121024LA) 010799 SC 12 μghIL-15 in ICFA (Difco, Lot no. 121195LA) 150799 SC 12 μg hIL-15 in ICFA020899 SC 12 μg hIL-15-KLH in ICFA 070999 SC 12 μg hIL-15-KLH in ICFA280999 SC 12 μg hIL-15-KLH in CFA 111099 IV 30 μg hIL-15 in PBS 121099IV 30 μg hIL-15 in PBS 151099 fusion of lymph node and spleen cells ofthis mouse with SP2/0

Mouse no. 404 (HCo7), ID 997-404, Female

201099 IP 25 μg hIL-15-KLH in CFA (Difco, lot no. 121024LA) 031199 IP12.5 μg hIL-15, 12.5 μg hIL-15-KLH, 25 μg in ICFA (Difco, lot no.121195LA) 101199 IV 12.5 μg hIL-15, 12.5 μg hIL-15-KLH 121199 IV 12.5 μghIL-15, 12.5 μg hIL-15-KLH 191199 fusion of lymph node and spleen cellsof this mouse with SP2/0

Culture Media

Fusion Partner Medium (FPM):

Iscoves Modified Dulbecco's Medium was supplemented with 100 IU/mlpenicillin, 100 μg/ml streptomycin, 1 mM Na-Pyruvate, 0.5 mMβ-mercaptoethanol (Life Technologies, Paisley, Scotland) and 10%heat-inactivated fetal calf serum (HyClone, Utah, USA).

Fusion Selection Medium (FSM):

FPM supplemented with 30 ml Origen Hybridoma Cloning Factor (IGEN,Gaithersburg, Md., USA), HAT (1 vial, manufacturer's recommendedconcentration, Sigma Chemical Co., St. Louis, Mo., USA) and 0.5 mg/mlkanamycin (Life Technologies, Paisley, Scotland).

Fusion Cloning Medium (FCM):

FPM supplemented with 20 ml Origen Hybridoma Cloning Factor (IGEN,Gaithersburg, Md., USA), HT (1 vial, manufacturer's recommendedconcentration, Sigma Chemical Co., St. Louis, Mo., USA) and 0.5 mg/mlkanamycin (Life Technologies, Paisley, Scotland).

Hybridoma Preparation: Fusion of Spleen and Lymph Node Cells with SP2/0Myeloma Cells

To obtain hybridomas, spleen, inguinal and para-aortic lymph nodes wereremoved from the mice. Single cells suspensions of spleen and lymph nodecells were mixed with SP2/0 myeloma cells in a cell ratio 1:2. Cellswere spun down and the pellet was resuspended gently in 1 mlpolyethyleneglycol (50% w/v in PBS, Sigma-Aldrich, Irvine, UK) at 37° C.After swirling the cells for 60 seconds, 25 ml FPM-2 was added and cellswere incubated at 37° C. for 30-60 minutes. After incubation, cells werecultured at a cell concentration of 0.75×10⁵ cells per well (in 100 μl)in 96-wells plates in FSM. After 3 days, 100 μl FSM was added to eachwell.

Fusion of spleen and lymph nodes of HCo7 and HCo12 mice immunised withhIL-15 resulted in the generation of several hybridomas producingantibodies directed against IL-15. The following four stable clonesproducing fully human anti-IL-15 antibodies were isolated: (1)146LyD7F7B7 renamed: 146B7; (2) 146DE2E12A3H5 renamed: 146H5; (3)404CG11B7E4 renamed: 404E4; and (4) 404FB12E7A8 renamed: 404A8. Theseclones were all of the human IgG1/k subclass.

Screening of the Hybridomas

Between day 7 and 11 after the fusion, the wells were screened for thepresence of human antibodies using the following ELISAs:

ELISA to Screen for the Presence of Human IgG in the CultureSupernatants

To perform the ELISA to detect the presence of human IgG antibodies, 100μg/well of 0.9 μg/ml rabbit-α-k-light chains antibodies (DAKO, Glostrup,Denmark) was added in phosphate buffered saline (PBS) to Nunc MaxisorpELISA-plate (incubation overnight at room temperature). After blockingthe plate with PBS supplemented with chicken serum (2%; LifeTechnologies, Paisley, Scotland) and Tween-20 (0.05%; PBSTC), culturesupernatants were added. After incubation for 1.5 hour the plates werewashed and rabbit-α-Human IgG (Fab2-fragments) conjugated with horseradish peroxidase (DAKO, Glostrup, Denmark) 0.5 μg/ml diluted in PBSTCwas added. After incubation for 1 hour, the wells were washed andsubstrate, ABTS (2,2′-Azinobis-3-ethylbenzthiazoline-sulphonic-acid,Roche Diagnostics, Mannheim, Germany) was added according to themanufacturer's protocol and antibody binding was evaluated at 405 nm inan EL808 ELISA-reader (Bio-tek Instruments, Winooski, Vt., USA).

ELISA to Screen for the Presence of IL-15 Specific Antibodies

Wells containing human IgG/k antibodies were further tested for thepresence of human anti-IL-15 antibodies in an IL-15-specific ELISA. Toperform the ELISA, 100 μl/well of 1 μg/ml IL-15 was added in phosphatebuffered saline (PBS) to Nunc Maxisorp ELISA-plate (incubation overnightat room temperature). After blocking the plate with PBS supplementedwith chicken serum (2%; Life Technologies, Paisley, Scotland) andTween-20 (0.05%; PBSTC), culture supernatants were added. Afterincubation for 1.5 hours the plates were washed and α-Human IgG Fcconjugated with horse radish peroxidase (Jackson immuno research, WestGrove, Pa., USA) 1/5000 diluted in PBSTC was added. After incubation for1 hour, the wells were washed and substrate, ABTS(2,2′-Azinobis-3-ethylbenzthiazoline-sulphonic-acid, Roche Diagnostics,Mannheim, Germany) was added according to the manufacturer's protocoland antibody binding was evaluated at 405 nm in an EL808 ELISA-reader(Bio-tek Instruments, Winooski, Vt., USA).

Subcloning of the Hybridomas

To obtain stable anti-IL-15 cell lines, the hybridomas were subcloned bya limiting dilution of the cells (to 0.5 cell/well) in 96-wells plates.

The subclones were tested after approximately 10 days with the abovementioned IL-15 ELISA. During the several subcloning procedures, FSM waschanged in phases via FCM to FPM. The isotype of the subclones wasdetermined with the ELISA described below.

Isotype Determination of the Anti-IL-15 Antibodies by ELISA

To perform the isotype ELISA, 100 μl/well of 1 μg/ml anti-human Fc(Jackson Immuno research) was added in phosphate buffered saline (PBS)to Nunc Maxisorp ELISA-plate (incubation overnight at room temperature).After blocking the plate with PBS supplemented with chicken serum (2%;Life Technologies, Paisley, Scotland) and Tween-20 (0.05%; PBSTC),culture supernatants were added. After incubation for 1.5 hours theplates were washed and mouse-α-HuIgG1 conjugated with alkalinephosphatase (Zymed, plates, land), or mouse-α-HuIgG3 conjugated withhorse radish peroxidase (Zymed) was added. After incubation for 1 hourthe wells were washed and substrate, ABTS(2,2′-Azinobis-3-ethylbenzthiazoline-sulphonic-acid, Roche Diagnostics,Mannheim, Germany) was added according to the manufacturer's protocol.Antibody binding was evaluated at 405 nm in an EL808 ELISA-reader(Bio-tek Instruments, Winooski, Vt., USA.

Example 4 Epitope Specificity of Fully Human Anti-IL-15 Antibodies

To function therapeutically and to inhibit IL-15-induced proinflammatoryeffects, IL-15 specific antibodies need to recognize the IL-15 epitopesinvolved in interaction with the IL-2Rβ-chain and/or the γ-chain ofIL-15 receptor.

Mutant proteins (described by Pettit et al.) were used to evaluate theepitope specificity of the fully human anti-IL-15 antibodies, 146B7,146H5, 404A8 and 404E4. The IL-15 mutants used include IL-15 mutantQ108S (Gln at residue 108 was replaced by Ser; a mutation in the γ-chaininteraction site) and mutant D8SQ108S (Gln at residue 108 was replacedby Ser and Asp at position 8 was substituted for Ser; mutations in boththe β and γ-chain interaction sites of IL-15).

ELISA to Determine Binding of the hIL-15 Specific Antibodies, 146B7,147H5, 404A8 and 404E4, to hIL-15 and to Mutant IL-15 Proteins

To perform the ELISA, 100 μl of 1 μg/ml IL-15 or hIL-15 mutant protein,in phosphate buffered saline (PBS), was added to Nunc MaxisorpELISA-plate for coating. After blocking the plate with PBS supplementedwith chicken serum (2%; Life Technologies, Paisley, Scotland) andTween-20 (0.05%; PBSTC), serial dilutions of the hIL-15 specificantibodies were incubated. After washing, α-Human IgG Fc conjugated withperoxidase (Jackson Immuno research, West Grove, Pa., USA) 1/5000diluted in PBSTC was added. After washing substrate, ABTS(2,2′-Azinobis-3-ethylbenzthiazoline-sulphonic-acid, Roche Diagnostics,Mannheim, Germany) was added according to the manufacturer's protocoland antibody binding was evaluated at 405 nm in an EL808 ELISA-reader(Bio-tek Instruments, Winooski, Vt., USA).

The binding of the fully human IL-15 specific antibodies 146B7, 146H5,404A8 and 404E4 to hIL-15 and to the IL-15 mutant proteins Q108S andD8SQ108S is shown in FIG. 1. Neither 146B7 nor 146H5 were able to bindto these mutant IL-15 proteins. Since both mutants carry the Q108Smutation, the epitope recognized by 146B7 and 146H5 is within thecritical domains of IL-15 which interact with the γ-chain of the IL-15receptor. 404A8 and 404E4 were both able to bind the mutant proteins,therefore, these antibodies recognize an epitope outside the β- andγ-chain interacting domains of IL-15. Both 146B7 and 146H5 bind to IL-15at the region that interacts with the γ-chain of the IL-15 receptor.This agrees with the data obtained from the proliferation assays usingthe fully human anti-IL-15 antibodies of the present invention. Asdescribed in detail below, neither 404A8 nor 404E4 were able to inhibitIL-15-induced proliferation of CTLL-2 cells and human PBMCs. Both 146B7and 146H5 were able to inhibit IL-15-induced proliferation. Further,inhibition of proliferation is achieved by blocking the interaction ofIL-15 with the γ-subunit of the IL-15 receptor.

Example 5 V_(H) and V_(L)—Region Sequences of 146B7

The nucleotide and deduced amino acid sequence of rearranged V_(H) andV_(L)-domains of 146B7 were determined using the following procedures.These sequences give information regarding the V_(H) and V_(L) germlinefamilies used; point mutations in these germline sequences are due toaffinity maturation of B-cells during the immunization of the animal.

RNA Preparation

Total RNA was prepared from 5×10⁶ 146B7 hybridoma cells with RNAzol(Biogenesis, Poole, England) according to the manufactures protocol.

cDNA Preparation

The cDNA of RNA from 146B7 was prepared from 3 μg total RNA with AMVReverse Transcriptase with buffer (Roche Diagnostics GmbH, Mannheim,Germany), oligo d(T)₁₅ (Promega, Madison, Wis., USA), dNTP (BoehringerMannheim corp., USA) and RNAsin (Promega) according to themanufacturer's protocol.

PCR Primers Used to Amplify V_(H) and V_(L) Regions for Cloning

Primer pairs used:

V_(H): FR1 5′ primers (1) AB62 CAg gTK CAg CTg gTg CAg TC (2) AB63 SAggTg CAg CTg KTg gAg TC (3) AB65 gAg gTg CAg CTg gTg CAg TC V_(H) leader5′ primers (4) AB85 ATg gAC Tgg ACC Tgg AgC ATC (5) AB86 ATg gAA TTg gggCTg AgC Tg (6) AB87 ATg gAg TTT ggR CTg AgC Tg (7) AB88 ATg AAA CAC CTgTgg TTC TTC (8) AB89 ATg ggg TCA ACC gCC ATC CT V_(H) 3′ primer (9) AB90TgC CAg ggg gAA gAC CgA Tgg VK: FR1 5′ primers (1) AB8 RAC ATC CAg ATgAYC CAg TC (2) AB9 gYC ATC YRg ATg ACC GAg TC (3) AB10 gAT ATT gTg ATgACC CAg AC (4) AB11 gAA ATT gTg TTg ACR CAg TC (5) AB12 gAA ATW gTR ATgACA CAg TC (6) AB13 gAT gTT gTg ATg ACA CAG TC (7) AB14 gAA ATT gTg CTgACT CAg TC V_(K) leader 5′ primers: (8) AB123 CCC gCT Cag CTC CTg gggCTC CTg (9) AB124 CCC TgC TCA gCT CCT ggg gCT gC (10) AB125 CCC AgC gCAgCT TCT CTT CCT CCT gC (11) AB126 ATg gAA CCA Tgg AAg CCC CAg CAC AgCV_(K) 3′ primer (12) AB16 Cgg gAA gAT gAA gAC AgA Tg

PCR Conditions Used to Amplify V_(H) and V_(L) Regions for Cloning

PCR Reactions were performed with AmpliTaq polymerase (Perkin Elmer) ona GeneAmp PCR System 9700 (Perkin Elmer Applied Biosystems, Foster City,Calif., USA).

PCR Cycling Protocol:

11 cycles 94° 2′ 94° 30″ 65° 30″, minus 1° per cycle 72° 30″ 30 cycles94° 30″ 55° 30″ 72° 30″ 72° 10′ cool down to 4°Cloning Of V_(H) and V_(L) in pGEMT-Vector System I

After analysing the PCR products on an agarose gel, the products werepurified with S-400 or S300 microspin columns (Amersham PharmaciaBiotech Inc., Piscataway, N.J., USA), or with the QIAEX II GelExtraction Kit (Qiagen GmbH, Hilden, Germany). For each experiment 2independently amplified PCR products, using FR1 or leader primers, ofeach V_(H) and V_(L) region were cloned in pGEMT-Vector System I(Promega) according to manufacturers protocol.

After transformation to E. coli DH5α, individual colonies were screenedby colony PCR using T7 and SP6 primers, 30 cycles at 55°. Plasmid DNAfrom each individual colony was purified using Qiaprep Spin miniprep kit(Qiagen). To further analyze a Nco1/Not1 (NE Biolabs, United Kingdom andRoche Diagnostics) digestion was performed and analyzed on agarose gel.

Sequencing

The V-regions were sequenced after cloning in the pGEMT-Vector System I.T7 and Sp6 primers (Eurogentec, Luik, Belgium) were used in combinationwith the sequence kit: ABI Prism BigDye Terminator Cycle SequencingReady Reaction Kit (Applied Biosystems, Warrington, United Kingdom)according to protocol. The reactions were performed on a ABI PRISM 377Sequencer (PE Applied Biosystems) and the sequences were analysed withthe program DNAStar, SeqmanII. The sequences were then aligned togermline V-gene sequences in VBASE(www.mrc-cpe.cam.ac.uk/imt-doc/public/intro.htm).

Cloning and Sequencing of the V_(H) and V_(L)-Region of 146B7

V_(H) and V_(L)-regions from hybridoma 146B7 were amplified by PCR andcloned in pGEMT-Vector System I to determine the cDNA-sequence. Thenucleotide and corresponding amino acid sequences are shown in FIG. 2(SEQ ID NOs: 1 and 2) and FIG. 3 (SEQ ID NOs: 3 and 4), respectively.The framework (FR) and complementarity determining regions (CDR) arealso indicated. The germline family for the V_(H)-region of 146B7according to alignment in Vbase: V_(H)5-51 (V_(H)5-subgroup), D2-15/D2(D_(H)-segment), JH4b (J_(H)-segment). The germline family for theV_(L)-region of 146B7 according to alignment in Vbase: A27(V_(K)III-subgroup) and J_(K)2 (J_(K)-segment). More informationregarding V_(H) and V_(L)-domains is shown at the Kabat databasehttp://immuno.bme.nwu.edu/ or at http://www.Vbase.com.

Example 6 Affinity Binding Characteristics of 146B7

The affinity of 146B7 was analyzed by surface plasmon resonance (SPR)technology using a BIACORE 3000 instrument to determine biomolecularprotein interactions according to the following procedures. Changes inthe SPR signal on the surface layer caused by biomolecular binding aredetected and signify a change in the mass concentration at the surfacelayer. Affinity is expressed using the following definitions:k_(a)=association rate constant (M⁻¹ sec⁻¹); k_(d)=dissociation rateconstant (sec⁻¹); K_(A)=association equilibrium constant=k_(a)/k_(d)(M⁻¹); and K_(D)=dissociation equilibrium constant=k_(d)/k_(a) (M).

Different procedures were performed to obtain the affinity of 146B7 forhuman IL-15 (hIL-15). Human recombinant IL-15 from two differentsuppliers (Immunex corp., Seattle, USA and Peprotech, Rocky Hill, N.J.,USA) was coupled to a CM5 sensor chip. The compound coupled to thesensorchip is defined as ligand. In other experiments 146B7 was used asligand.

In each kinetic analysis, the binding of the analyte, 146B7 or hIL-15adapted to the ligand coupled to the sensorchip, was compared to thebinding to a reference control CM5 sensor chip. Serial dilutions ofanalyte were tested (0, 3.125, 6.25, 12.5, 25, 50 μg/ml). Associationand dissociation curves were fitted for monomeric interaction in themodel Langmuir 1:1, to determine k_(a) and k_(d) and to calculate K_(A)and K_(D). All data were analyzed using BIA-Evaluation Version 3.1. Fora bivalent interaction the model “bivalent analyte” was used. Allanalysis were corrected for a drifting baseline.

To determine the antibody affinity of 146B7, the affinity of antibody146B7 was measured for human recombinant IL-15 derived from twodifferent suppliers, Immunex and Peprotech, at the BIACORE 3000. Using146B7 as ligand and hIL-15 as analyte, the monovalent interaction wasdetermined (curve fitting Langmuir 1:1).

The affinity of 146B7 for IL-15 (Immunex Corp.) was measured as follows:

The association rate constant k_(a): 1.07 (±0.17) × 10⁵ M⁻¹ sec⁻¹ Thedissociation rate constant k_(d): 6.56 (±0.09) × 10⁻³ sec⁻¹ Associationequilibrium constant K_(A): 1.55 (±0.21) × 10⁷ M⁻¹ Dissociationequilibrium constant K_(D): 6.59 (±0.88) × 10⁻⁸ M

To determine the avidity of 146B7, IL-15 (Immunex Corp.) was used asligand and 146B7 was used as analyte. When the data obtained wereanalyzed using Langmuir (1:1) curve fitting the bivalent interaction ofthe antibody was expressed, the avidity of the antibody was determined.

The avidity of 146B7 for IL-15 (Immunex Corp.) was measured as follows:

The association rate constant k_(a): 7.30 (±0.81) × 10⁵ M⁻¹ sec⁻¹ Thedissociation rate constant k_(d): 1.45 (±2.05) × 10⁻³ sec⁻¹ Associationequilibrium constant K_(A): 5.03 (±3.40) × 10⁸ M⁻¹ Dissociationequilibrium constant K_(D): 1.55 (±1.24) × 10⁻⁹ M

The affinity and avidity of 146B7 for Peprotech derived IL-15 weredetermined also. No major differences in affinity or avidity for twodifferent sources of IL-15 were seen.

As is described in the example below regarding the inhibition of humaninterleukin-15 (hIL-15)-induced proliferation of CTLL-2 cells and PBMCby fully human anti-IL-15 antibodies, 146B7 inhibited in a dosedependent manner the IL-15 induced proliferation as was measured by[³H]-thymidine incorporation. The IC₅₀—concentration at 50% inhibition,a more functional manner to determine affinity—from these proliferationinhibition experiments was calculated: 3.1±0.91 nM. This IC50 is inagreement with the avidity measured by BIACORE 3000 (K_(D) 1.5 nM) using146B7 as ligand and recombinant human IL-15 as analyte and confirmed theaffinity and avidity measurements obtained here.

Example 7 Inhibition of hIL-15-Induced TNF-α Production by Fully HumanAnti-IL-15 Antibodies

The effect of fully human anti-IL-15 antibodies, 146B7, 146H5, 404E4 and404A8, on IL-15-induced TNF-α production was studied using peripheralblood derived mononuclear cells (PBMC) from healthy volunteers using thefollowing procedures. To evaluate specificity to IL-15, the effect ofthese antibodies on IL-2-mediated TNF-α production was also examined.

Cell Culture

Cultures were maintained in RPMI-1640 with 2 mM L-glutamine, 100 IU/mlpenicillin, 100 μg/ml streptomycin (all derived from Life Technologies,Paisley, Scotland) and 10% heat-inactivated fetal calf serum (HyClone,Utah, USA).

Purification of Peripheral Blood Mononuclear Cells (PBMC)

Fresh human blood was drawn from a healthy volunteer after informedconsent, heparin was added against coagulation. Purification of PBMC wasperformed by density gradient centrifugation using Ficoll (Pharmacia,Uppsala, Sweden).

Test Compound

HIL-15, lot no: 6870-011, Immunex corp., Seattle, Wash., USA.

hIL-2, Chiron Benelux BV, Amsterdam, The Netherlands.

Fully human antibodies used: 146B7 (batch: 070101) and 146B7RDJW07,404A8 (batch: 030101) and 404E4 (batch: 080101) and as isotype controlantibody T1 (97-2B11-2B12, batch: 190900).

Inhibition of Human IL-15 (hIL-15) or hIL-2-Induced TNF-α Production byPBMC by Anti-IL-15 Antibodies

PBMC were cultured in triplicate or quadruplicate in 96-well flat-bottomplate at 1.5×10⁵ cells per well in the presence or absence of hIL-2 orhIL-15 and with or without anti-IL-15 antibodies. Isotype controlantibody (T1) was included as negative control. Concanavalin A (2.5μg/ml, Calbiochem) was added as a positive control for proliferation.Cells were incubated for 72 hours at 37° C. and 5% CO₂. Supernatantswere harvested to quantify the amount of human TNF-α by ELISA (U-CyTech,Utrecht, The Netherlands).

The effects of 146B7 and an isotype control antibody were tested onIL-15-mediated TNF-α production by PBMC. 146B7 inhibited hIL-15-mediatedTNF-α production in a dose dependent fashion, whereas the isotypecontrol antibody did not inhibit hIL-15-induced TNF-α production (FIG.4). Data of two healthy volunteers are shown. 404E4 and 404A8 wereunable to inhibit hIL-15-induced TNF-α production.

To ensure the specificity of the anti-IL-15 antibodies, their effect onhIL-2-mediated TNF-α production was evaluated. No inhibition ofIL-2-mediated TNF-α production was induced by 146B7 (FIG. 5). No dosedependent inhibition by either 404E4 or 404A8 was seen in hIL-2-mediatedTNF-α production.

A dose dependent inhibition of hIL-15-mediated TNF-α production was seenonly by 146B7 and not by 404E4 and 404A8. The inhibitory effect wasspecific for hIL-15; IL-2-mediated TNF-α production was not inhibited.

Example 8 Inhibition of Human Interleukin-15 (hIL-15)-InducedProliferation of CTLL-2 Cells and PBMC by Fully Human Anti-IL-15Antibodies

Antibodies 146B7, 146H5, 404E4 and 404A8 were tested for their abilityto inhibit T-cell proliferation using CTLL-2 cells (Gillis et al., 1978)and peripheral blood mononuclear cells (PBMC) using the followingprocedures.

Cell Culture

Cultures were maintained in RPMI-1640 with 2 mM L-glutamine, 100 IU/mlpenicillin, 100 μg/ml streptomycin (derived from Life Technologies,Paisley, Scotland) and 10% heat-inactivated fetal calf serum (HyClone,Utah, USA). CTLL-2 cells (Gillis et al., 1978) were maintained in theabove mentioned medium supplemented with 36 units hIL-2/ml (ChironBenelux BV, Amsterdam, The Netherlands) and starved for hIL-2 for 3-4days before the start of the experiment. CTLL-2 cells were washed threetimes before use.

Purification of Peripheral Blood Mononuclear Cells (PBMC)

Fresh human blood was drawn from a healthy volunteer after informedconsent, heparin was added against coagulation. Purification of PBMC wasperformed by density gradient centrifugation using Ficoll (Pharmacia,Uppsala, Sweden).

Test Compound

HIL-15, lot no: 6870-011, Immunex corp., Seattle, Wash., USA.

hIL-2, Chiron Benelux BV, Amsterdam, The Netherlands.

anti-IL-15 antibodies used for CTLL-2 assay in this report shown in FIG.6: 146B7, 146H5, 404A8, 404E4.

anti-IL-15 antibodies used for PBMC assays: 146B7 (batch: 070101), 404A8(batch: 030101) and 404E4 (batch: 080101).

Inhibition of Human IL-15 (hIL-15) or hIL-2 Induced CTLL-2 Proliferationby Anti-IL-15 Antibodies

In each experiment, cells were seeded in triplicate in 96-well plate,5×10³ cells per well in the presence or absence of either hIL-2 orhIL-15. To evaluate the effect on proliferation, each of the fouranti-IL-15 antibodies were added. Cells were incubated for 16 hours at37° C. and 5% CO₂. [³H]Thymidine (1 μCi/well, Amersham Life Sciences,Little Chalfont, Buckinhamshire, UK) was added 4 hours before harvesting(Harvester 96 Mach II M, Tomtec, Orange Conn., USA).

As is shown in FIG. 6, IL-15 induced proliferation of CTLL-2 cells wasdecreased in a dose dependent fashion by 146B7 and 146H5 as wasreflected by reduced [³H]-Thymidine incorporation. Both 404E4 and 404A8were unable to block IL-15 induced proliferation of CTLL-2 cells.

Inhibition of hIL-15 (hIL-15) or hIL-2 Induced PBMC Proliferation byAnti-IL-15 Antibodies

PBMC were cultured in triplicate in 96-well U-bottom plate (Nunc, NalgeNunc International, Denmark), 5×10⁴ cells per well in the presence orabsence of hIL-2 or hIL-15 and the anti-IL-15 antibodies. Concanavalin A(2.5 μg/ml, Calbiochem) was added as a positive control forproliferation. The cells were incubated for 72 hours at 37° C. and 5%CO₂. [3H]Thymidine (1 μCi/well, Amersham Life Sciences, Little Chalfont,Buckinhamshire, UK) was added 16 hours before harvesting (Harvester 96,Tomtec, Orange Conn., USA).

146B7 was able to inhibit IL-15 induced [³H]-Thymidine incorporationdose dependently and, therefore, inhibited proliferation (IC50=3.1±0.91nM) (FIG. 7). Both 404E4 and 404A8 were unable to block hIL-15 inducedPBMC proliferation (FIGS. 8-9). 146H5 was not tested according to dataobtained from previously performed experiments.

To ensure the specificity of 146B7, 404E4 and 404A8 for IL-15, theseantibodies were also evaluated for their effects on IL-2 mediatedproliferation. None of the tested anti-IL-15 antibodies exhibited aneffect on IL-2 induced proliferation (FIGS. 7-9).

Example 9 Human Anti-IL-15 Antibody 146B7 Binds to Human IL-15 Presenton Human PBMCs Test Compounds

Human PBMCs were obtained from healthy volunteers after informedconsent.

Antibody 146B7 (batch no. MDX015), Medarex Inc., Annandale, N.J., USA.

Biotinylation of 146B7 and Human IgG

N-hydroxysuccinimido-biotin (Sigma) was first diluted in DMSO (finaldilution: 100 mg/ml) and then in 0.1 M NaHCO₃ (final dilution: 1 mg/ml,Sigma). Per 1 mg of antibody (diluted in 1 ml), 600 μl of biotinsolution was added (dark, 2 hrs, RT). Antibody-biotin solution wasdialysed in a Slide-a-lyzer™ dialysis cassette (10,000 MWCO, Pierce,Perbio Science, Netherlands) (overnight at 4° C.) to remove unlabeledbiotin. The following day, concentration of biotinylated antibodies wasdetermined by spectrophotometry (Ultrospec 2100pro) at OD 280 nm.

Stimulation of Peripheral Blood

To induce IL-15, blood was obtained by venapuncture from healthyvolunteers. PBMCs were cultured in RPMI 1640 (Biowhittaker Europe)supplemented with penicillin (5 U/ml), streptomycin (50 μg/ml),L-glutamine (2 mM) (Biowhittaker Europe), and 10% fetal calf serum(Optimum C241, Multicell, Wisent Inc.) for a maximum of 2 days (37° C.),and were stimulated with 500 U/ml IFNγ (Boehringer Ingelheim).

Flow Cytometry

Cells were pre-incubated with 10% human AB serum (CLB, Amsterdam,Netherlands) in RPMI 1640 (Biowhittaker Europe) supplemented withpenicillin (5 U/ml), streptomycin (50 μg/ml), L-glutamine (2 mM)(Biowhittaker Europe) and 10% fetal calf serum (Optimum C241, Multicell,Wisent Inc.). After permeabilization (20 min, 4° C., inCytofix/Cytoperm™ Kit, Becton Dickinson, San Diego, Calif.) and washingin Perm/Wash™ buffer (Cytofix/Cytoperm™ Kit), PBMC were subjected tostaining of IL-15 by flow cytometry. Continuous permeability wasachieved by using Perm/Wash™ buffer (Cytofix/Cytoperm™ Kit) throughoutthe staining procedure. After incubating the cells with biotinylated146B7 or with biotinylated hIgG1 (20 μg/ml, 30 min, 4° C.) and washingin Perm/Wash™ buffer, cells were subsequently incubated withstreptavidin-phycoerythrin (DAKO) for 30 minutes (4° C.). Fluorescenceintensity of at least 5000 cells per sample was determined afteranalysis by flow cytometry (FACS Calibur, Becton Dickinson) and gatingon the monocytes, using CellQuest Pro software. Data show thestimulation index (S.I.), which is calculated as follows: S.I.=(meanfluorescence positive staining)/(mean fluorescence background staining)

Immunocytochemistry

To detect IL-15 present in human monocytes, cytospin preparations weremade of whole blood samples. After spinning down 5×10⁴ cells (200 μl)onto Superfrost®-Plus microscope slides (Menzel), slides were air-dried(<60 min), fixed in 2% paraformaldehyde/PBS (8 min, 4° C.), washed withPBS and air-dried again. Before staining, cytospin preparations werepermeabilized in PBS (+0.1% saponine; PBSS), which was subsequently usedthroughout the staining procedure. To block endogenous peroxidaseactivity, cytospin preparations were incubated with 0.05% (v/v) hydrogenperoxide (H₂O₂) diluted in citric acid/phosphate buffer (pH 5.8, 20 min,RT). After washing with PBSS, endogenous biotin activity was blockedaccording to the manufacturer's instructions (Biotin Blocking Kit,Vector Lab., DAKO). After washing with PBSS, non-specific binding siteswere blocked by incubating the cytospin preparations with 10% (v/v)human pooled AB-serum (CLB, Amsterdam, Netherlands) (30 min) in PBSS.Thereafter, cytospin preparations were incubated with biotinylatedprimary antibody (60 min, RT) and, after washing with PBSS, withstreptavidin complexed with biotinylated horseradish peroxidase(streptABComplex/HRP, DAKO; 1:100 in PBSS, containing 2% human AB serum;30 min, RT). After washing in PBSS, the cytospin preparations wereincubated with 3-amino-9-ethylcarbazole (0.5 mg/ml) and H₂O₂ (0.01%), insodium acetate buffer (50 mM, pH 4.9) for 10 minutes (RT), for thedetection of HRP activity. Cytospins were washed with running tap waterfor 5 minutes, counterstained with haematoxylin (DAKO) for one minute,washed with running tap water for another 5 minutes, and embedded infaramount or glycergel (DAKO).

Flow Cytometry

Binding of 146B7 to IFNγ-stimulated human monocytes is shown in FIG. 10.Biotinylated 146B7 binds to unstimulated monocytes showing the presenceof IL-15 in unstimulated cells. Stimulation of monocytes with IFNγ leadsto a increased binding of 146B7 to the cells, with a maximum reached atday one of culture. The control antibody, hIgG1, shows little binding tounstimulated monocytes. Stimulation with IFNγ increases binding of hIgG1through increased expression of Fcγ receptors on monocytes.

Immunocytochemistry

FIG. 11 shows staining of human monocytes with 146B7, or with thecontrol antibody, hIgG1. A clear red staining of the cytoplasm isobserved after incubating the cells with 146B7, but not with the controlantibody. Accordingly, 146B7 binds hIL-15 in monocytes and this bindingis upregulated after stimulation with IFNγ. FIG. 11 also shows thatIL-15 staining is primarily intracellular.

Example 10 Human Anti-IL-15 Antibody 146B7 Binds IL-15 in Tissues byImmunohistochemistry Test Compounds

Human psoriatic skin-tissue samples were obtained after informedconsent. Louise Villadsen, Department of Dermatology, GentofteUniversity Hospital, Copenhagen, Denmark.

Antibody 146B7 (batch no. MDX015), Medarex, Annandale, N.J., USA

Biotinylation of 146B7 and Human IgG

N-hydroxysuccinimido-biotin (Sigma) was first diluted in DMSO (finaldilution: 100 mg/ml) and then in 0.1 M NaHCO₃ (final dilution: 1 mg/ml,Sigma). Per 1 mg of antibody (diluted in 1 ml), 600 μl of biotinsolution was added (dark, 2 hrs, RT). Antibody-biotin solution wasdialysed in a Slide-a-lyzer™ dialysis cassette (10,000 MWCO, Pierce,Perbio Science, Netherlands) (ON, 4° C.) to remove unlabeled biotin. Thefollowing day, concentration of biotinylated antibodies was determinedby spectrophotometry (Ultrospec 2100pro) at OD 280 nm.

Immunohistochemistry

Tissues were stored at −80° C. until assay. After thawing, tissuesections were fixated in acetone (10 min, RT) and air-dried. To blockendogenous peroxidase activity, sections were incubated with 0.05% (v/v)hydrogen peroxide (H₂O₂) diluted in citric acid/phosphate buffer (pH5.8, 20 min, RT). After washing with PBS-Tween 20 (PBST, 0.05% v/v),endogenous biotin activity was blocked according to the manufacturer'sinstructions (Biotin Blocking Kit, Vector Lab., DAKO). After washingwith PBST, non-specific binding sites were blocked by incubating thetissue sections with 10% (v/v) human pooled AB-serum (CLB, Amsterdam,Netherlands) (30 min) in PBST. Serum was blotted off and sections weresubsequently incubated with biotinylated primary antibody (146B7 orhIgG1) diluted in PBS containing 2% human AB serum for 60 minutes (RT).Sections were washed in PBST. After washing in PBST, all tissue sectionswere incubated with streptABComplex/HRP (DAKO; 1:100 diluted in PBScontaining 2% human AB serum; 30 min, RT). After washing in PBST, thesections were incubated with 3-amino-9-ethylcarbazole (0.5 mg/ml) andH₂O₂ (0.01%), in sodium acetate buffer (50 mM, pH 4.9) for 10 minutes(RT), for the detection of HRP activity. Sections were washed withrunning tap water for 5 minutes, counterstained with haematoxylin (DAKO)for one minute, washed with running tap water for another 5 minutes, andfinally embedded in faramount or glycergel (DAKO).

Results

A clear cytoplasmic staining of keratinocytes in psoriatic skin wasobserved after staining tissue sections with 146B7, but not with thecontrol antibody (FIG. 12; 146B7 stains IL-15-positive keratinocytesobtained from psoriatic plaques).

Example 11 Human Anti-IL-15 Antibody 146B7 Blocks IL-15 in SCIDMouse-Human Tissue Chimeras: Significant Inhibition of Inflammation inBoth Arthritic and Psoriatic Tissue Test Compounds

Synovial tissue—obtained form patients with juvenile rheumatoidarthritis, after informed consent; Alexei Grom, division of pediatricrheumatology, Children's Hospital Medical Center, Cincinnati, Ohio, USA.

Keratome biopsies—tissue samples were obtained after informed consent.Louise Villadsen, Department of Dermatology, Gentofte UniversityHospital, Copenhagen, Denmark.

Antibody 146B7 (batch no. MDX015), Medarex Inc., Annandale, N.J., USAfor psoriasis experiments.

Antibody 146B7 (batch no. 15-00RDJW07), Medarex Inc., Annandale, N.J.,USA for rheumatoid arthritis experiments.

Blocking IL-15 in SCID Mouse-Human Synovial Tissue Chimeras

Fresh synovial tissue samples were obtained from patients with juvenilerheumatoid arthritis after joint replacement surgery. Samples werecollected in sterile conditions. Minced tissue fragments from the entiresynovial tissue sample were thoroughly mixed to ensure homogeneity ofeach preparation. Minced tissues (2-4 grafts per animal; 100 mg per onesite) were engrafted subcutaneously in the back of SCID/NOD mice(Jackson Laboratories). Each animal received 146B7 (500 μg, i.p.) or PBSon the day of graft implantation, and on post-implantation days 7, 14,and 21. Animals were sacrificed on day 28 post-implantation. Synovialgrafts were excised and placed on formalin for H&E staining.

Quantification of H&E Staining of Tissues from SCID Mouse-Human SynovialTissue Chimeras (Modified from Lehr et al., J. Histochem. Cytochem.1997, 45, 1559)

After obtaining digital images (2600×2060, jpg) of sections obtainedfrom SCID mouse-human synovial tissue chimeras using a X10 objective(Zeiss microscope; Axiovision software), data were computer-analysed, byuse of Photoshop, version 6.0 (Adobe Systems, Mountain view, Calif.) andreduced to 1300×1300 pixels. Within each section six X10 fields werechosen so as to best reflect the overall staining of the tissue on theentire slide. After selection of all stained nuclei (magic wand on darknucleus with tolerance 10), an optical density plot of the selected areawas generated and the mean staining intensity was recorded (afterselection of similar/image histogram command). Subsequently, thebackground was selected and staining was quantified (magic wand onbackground with tolerance 10). Staining intensity was calculated as thedifference between nuclear staining and background staining. This wasdesignated the cytochemical index with arbitrary units. Data are shownas mean and s.e.m. Data were analysed by Student's t-test.

Blocking IL-15 in SCID Mouse-Human Psoriatic Tissue Chimeras

Keratome biopsies were obtained from psoriatic plaques of two patients,divided and transplanted onto C.B-17 SCID (Jackson Laboratories) mice.Three weeks after transplantation mice received PBS (placebo), CsA(cyclosporine A) (Sandoz) at a dose of 10 mg/kg every second day for 15days, or 146B7 at a dose of 20 mg/kg on day 1 and 10 mg/kg on days 8 and15. One week after the last injection, mice were sacrificed, and a 4 mmpunch biopsy was taken from each xenograft. Biopsies were fixed informalin for paraffin embedding and stained in H&E (FIG. 15) and forKi-67 nuclear antigen (FIG. 16).

Quantification of Immunohistochemical Staining of Tissues from SCIDMouse-Human Psoriatic Tissue Chimeras

The H&E-stained sections were evaluated for epidermal thickness (μm),grade of parakeratosis (rated from 0-3), and number of inflammatorymononuclear cells in upper dermis. The sections stained for Ki-67 wereevaluated for number of cycling keratinocytes/mm² section. Mean valuesfor the 4 mice in each treatment group were calculated, and the datafrom each patient were summarised as mean and s.e.m.

SCID/RA Model

Microscopic observation of sections showed that the darkest stainednuclei belong to infiltrating cells. Therefore, the number of nuclei(measured as the relative surface area) are considered as a measure forinfiltration. Injection of 146B7 reduces the number of infiltratingcells into inflamed synovial tissue, as compared to vehicle treatment(FIG. 13 a, p<0.05). FIGS. 13B and 13C illustrate the effects of 146B7(FIG. 13C) on infiltration of cells into xenografted synovial tissue,and show a reduction in number of cells with dark nuclei, as compared tovehicle treatment (FIG. 13B).

SCID/Psoriasis Model

FIG. 14 shows SCID/psoriasis mice treated with 146B7 or controltreatment. Compared to the vehicle, PBS, injections of 146B7 reduced theseverity of psoriasis evaluated by epidermal thickness when was measuredfrom the stratum corneum to the beginning of the rete pegs (FIG. 14A):PBS (177.8^(±)42.2 μm), CsA (91.0^(±)15.2 μm), 146B7 (62.5^(±)9.1 μm). Areduction in thickness was also observed when was measured from thestratum corneum to the deepest part of the rete pegs (FIG. 14B): PBS(433.8^(±)32.1 μm), CsA (303.8^(±)62.9 μm) and 146B7 (208.0^(±)33.8 μm).Also, the grade of parakeratosis was reduced by 146B7 treatment (FIG.14C): PBS (1.6^(±)0.4), CsA (1.3^(±)0.3), 146B7 (0.5^(±)0.3).Furthermore, 146B7 reduces the number of inflammatory mononuclear cellsin upper dermis (FIG. 14D): PBS (33.3-1.9 mononuclear cells), CsA(19.4^(±)8.5), 146B7 (16.4^(±)0.1). The expression of the human Ki-67protein is strictly associated with cell proliferation. Duringinterphase, the antigen can be exclusively detected within the nucleus,whereas in mitosis most of the protein is relocated to the surface ofthe chromosomes. The fact that the Ki-67 protein is present during allactive phases of the cell cycle (G(1), S, G(2), and mitosis), but isabsent from resting cells (G(0)), makes it an excellent marker fordetermining the so-called growth fraction of a given cell population.146B7 reduces the number of Ki-67+ cycling keratinocytes (FIG. 14E): PBS(247.9±77.0), CsA (116.0±24.1), 146B7 (73.8±9.9).

Treatment with 146B7 inhibited the infiltration of inflammatory cellsinto inflamed tissue in human SCID models for rheumatoid arthritis.Furthermore, in SCID mice with engrafted human psoriatic plaques,treatment with 146B7 reduced the severity of psoriasis, as compared totreatment with CsA. Indeed, treatment with 146B7 resulted in a majorreduction in inflammation, in epidermal thickness, in numbers ofdividing keratinocytes, and in severity of parakeratosis in human/SCIDmice.

Example 12 Human Anti-IL-15 Antibody 146B7 Recognizes Receptor-BoundIL-15 Test Compounds

hIgG1-human control antibody (Sigma).

Antibody 146B7-Medarex Inc., Annandale, N.J., USA, MDX015.

Raji cells with constitutive expression of IL-15Rα (Martin Glennie,Tenovus Research Laboratory, Southampton General Hospital, Southampton,U.K.).

Biotinylation of 146B7 and Human IgG

N-hydroxysuccinimido-biotin (Sigma) was first diluted in DMSO (finaldilution: 100 mg/ml) and then in 0.1 M NaHCO₃ (final dilution: 1 mg/ml,Sigma). Per 1 mg of antibody (diluted in 1 ml), 600 μl of biotinsolution was added (dark, 2 hrs, RT). Antibody-biotin solution wasdialysed in a Slide-a-lyzer™ dialysis cassette (10,000 MWCO, Pierce,Perbio Science, Netherlands) (overnight at 4° C.) to remove unlabeledbiotin. The following day, concentration of biotinylated antibodies wasdetermined by spectrophotometry (Ultrospec 2100pro) at OD 280 nm.

Binding of 146B7 to IL-15-IL-15Rα Complex by ELISA

After coating (overnight at room temperature) flat bottom microtiterplates (Greiner) with IL-15Rα (R&D systems, Minneapolis, Minn., USA),plates were incubated with PBS and chicken serum (2%, RT, 60 min). Afterwashing in PBS (+0.05% Tween 20: PBST), plates were subsequentlyincubated with several dilutions of unlabeled IL-15 (50 μl, RT, Immunex,Seattle, USA). After 10 minutes, biotinylated antibodies were added tothe wells (50 μl) in different concentrations (90 minutes at roomtemperature). After washing in PBST, plates were incubated (60 minutesat room temperature) with streptavidin-poly-horseradish peroxidase (CLB,Amsterdam, Netherlands) diluted 1:10,000 in PBST-C (PBST and 2% chickenserum). Finally, plates were washed and subsequently incubated with ABTS(Azinobis-3-ethylbenzthiazoline-sulphonic-acid, Roche Diagnostics,Mannheim, Germany) in ABTS buffer according to the manufacturer'sprotocol. Color reaction was stopped with 2% oxalic acid (50 μl).Binding was evaluated at 405 nm in an EL808 ELISA-reader (Bio-TekInstruments, Winooski, Vt., USA).

Binding of 146B7 to IL-15-IL-15R Complex on Raji Cells

Raji cells are pre-incubated (20 minutes at 4° C.) with 10% human pooledAB serum (CLB, Amsterdam, Netherlands) in FACS buffer (PBS, 0.05% BSA,0.02% NaNO₃). Raji cells (1-2*10⁵ cells/ml) were put in the wells, and50 μl of unlabeled IL-15 was added in several concentrations (diluted inFACS buffer with 10% human AB serum). After incubating the cells for 30minutes (4° C.) and washing twice in FACS buffer, 50 μl of biotinylatedantibodies (146B7 or hIgG1) was added to the wells (30 minutes at 4°C.). After washing twice in FACS buffer, 50 μl ofstreptavidin-phycoerythrin was added to each well (30 minutes at 4° C.).After washing twice in FACS buffer, cells were taken up in 200 μl ofFACS buffer, and fluorescence intensity of at least 5000 cells persample was determined after analysis by flow cytometry (FACS Calibur,Becton Dickinson) using CellQuest software. Data show the stimulationindex (S.I.), which is calculated as follows:

S.I.=(mean fluorescence positive staining)/(mean fluorescence backgroundstaining)

ELISA

Binding of 146B7 to IL-15/IL-15R complex in ELISA is shown in FIG. 17.Binding of 146B7 increases with increasing concentrations of IL-15binding to its receptor. No effects were observed of binding of controlantibody to IL-15 or to IL-15R.

Binding to IL-15R-Expressing Raji Cells

Binding of 146B7 to IL-15/IL-15R complex on Raji cells is shown in FIG.18. 146B7 binds to the IL-15/IL-15R complex in a dose-dependent manner.No binding of hIgG1 to the IL-15/IL-15R complex on Raji cells wasobserved (FIG. 18).

146B7 is able to bind IL-15 after binding of this cytokine to itsreceptor. 146B7 binds to an epitope on IL-15 that is not involved inbinding to the receptor.

REFERENCES

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. Any combination ofthe embodiments disclosed in the dependent claims are contemplated to bewithin the scope of the invention.

INCORPORATION BY REFERENCE

All publications, patents, and pending patent applications referred toherein are hereby incorporated by reference in their entirety.

1. An isolated nucleic acid encoding a heavy chain variable region of anantibody, wherein the variable region comprises the amino acid sequenceset forth in SEQ ID NO:2, or an amino acid sequence at least 80%identical thereto, and wherein the antibody binds to human IL-15.
 2. Anisolated nucleic acid encoding a light chain variable region of anantibody, wherein the variable region comprises the amino acid sequenceset forth in SEQ ID NO:4, or an amino acid sequence at least 80%identical thereto, and wherein the antibody binds to human IL-15.
 3. Anisolated nucleic acid encoding a heavy chain variable region of anantibody, wherein the nucleic acid comprises the nucleotide sequence setforth in SEQ ID NO:1, or a nucleotide sequence at least 80% identicalthereto, and wherein the antibody binds to human IL-15.
 4. An isolatednucleic acid encoding a light chain variable region of an antibody,wherein the nucleic acid comprises the nucleotide sequence set forth inSEQ ID NO:3, or a nucleotide sequence at least 80% identical thereto,and wherein the antibody binds to human IL-15.
 5. An isolated nucleicacid encoding a variable region of an antibody, wherein the antibodycomprises a heavy and light chain variable region having the amino acidsequences set forth in SEQ ID NOs:2 and 4, respectively, or amino acidsequences at least 80% identical thereto, and wherein the antibody bindsto human IL-15.
 6. An isolated nucleic acid encoding a variable regionof an antibody, wherein the antibody comprises a heavy and light chainvariable region encoded by the nucleotide sequences set forth in SEQ IDNOs:1 and 3, respectively, or nucleotide sequences at least 80%identical thereto, and wherein the antibody binds to human IL-15.
 7. Anexpression vector comprising the nucleic acid set forth in claim 1 or 2.8. An isolated nucleic acid encoding a variable region of an antibodythat binds an epitope on human IL-15 recognized by an antibodycomprising heavy chain and human light chain variable regions comprisingthe amino acid sequences set forth in SEQ ID NO:2 and SEQ ID NO:4,respectively.
 9. An isolated nucleic acid encoding a heavy chainvariable region of an antibody, wherein the variable region comprisesCDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs:5, 6, and 7,respectively, and wherein the antibody binds human IL-15.
 10. Anisolated nucleic acid encoding a light chain variable region of anantibody, wherein the variable region comprises CDR1, CDR2, and CDR3sequences as shown in SEQ ID NOs:8, 9, and 10, respectively, and whereinthe antibody binds human IL-15.
 11. An isolated nucleic acid encoding avariable region of an antibody, wherein the antibody binds human IL-15and comprises (a) a heavy chain variable region comprising CDR1, CDR2,and CDR3 sequences as shown in SEQ ID NOs:5, 6, and 7, respectively; and(b) a light chain variable region comprising CDR1, CDR2, and CDR3sequences as shown in SEQ ID NOs:8, 9, and 10, respectively.
 12. Anexpression vector comprising the nucleic acid set forth in claim 9 or10.
 13. The nucleic acid of claim 8, wherein an antibody inhibitsIL-15-induced proinflammatory effects.
 14. The nucleic acid of claim 13,wherein the antibody inhibits IL-15-induced TNFα production or T cellproliferation.
 15. The nucleic acid of claim 8, wherein the antibodybinds to human IL-15 with a dissociation equilibrium constant (K_(D)) ofbelow 10⁻⁷ M as determined by surface plasmon resonance (SPR) technologyusing recombinant human IL-15 as the analyte and the antibody as theligand.
 16. The nucleic acid of claim 8, wherein the antibody binds toan epitope located on the β-chain or the γ-chain interacting domain ofhuman IL-15.
 17. The nucleic acid of claim 8, wherein the antibody bindsto receptor-bound human IL-15.
 18. The nucleic acid of claim 8, whereinthe antibody is selected from the group consisting of an IgG1, IgG2,IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE antibody.
 19. Thenucleic acid of claim 18, wherein the antibody is an IgG1 antibody. 20.The nucleic acid of claim 18, wherein the antibody comprises a variableregion from an IgG1 heavy chain and a variable region from a kappa lightchain.
 21. The nucleic acid of claim 8, wherein the antibody is a Fabfragment or a single chain antibody.
 22. The nucleic acid of claim 8,wherein the antibody is produced by a hybridoma comprising a B cellobtained from a transgenic non-human animal having a genome comprising ahuman heavy chain transgene and a human light chain transgene fused toan immortalized cell.
 23. A transfectoma comprising the expressionvector of claim
 7. 24. A host cell comprising the expression vector ofclaim 23.